Complement Factor B Analogs and Their Uses

ABSTRACT

The invention provides polypeptides comprising a complement factor B analog. The invention also provides various complement factor B analogs including complement factor B analogs comprising a mutation of a free cysteine amino acid and related methods, nucleic acids and vectors. These complement factor B analogs and related methods, nucleic acids and vectors can be used to modulate a complement pathway or for the study and/or treatment of various conditions or diseases related to a complement pathway.

BACKGROUND OF THE INVENTION

The complement system is a component of the innate and adaptive immunesystem (reviewed by Volanakis, J. E., 1998. Chapter 2. In The HumanComplement System in Health and Disease. Edited by J. E. Volanakis, andM. M. Frank. Marcel Dekker, Inc., New York pp 9-32). Complement plays animportant role in microbial killing, and for the transport and clearanceof immune complexes. Many of the activation products of the complementsystem are also associated with proinflammatory or immunoregulatoryfunctions. The complement system consists of plasma andmembrane-associated proteins that are organized in threeenzymatic-activation cascades: the classical, the lectin, and thealternative pathways (FIG. 1). All three pathways can lead to theformation of the terminal complement complex (TCC) and an array ofbiologically active products.

In some cases, complement activation is initiated either by specificantibodies recognizing and binding to a variety of pathogens and foreignmolecules, and/or by direct interaction of complement proteins withforeign substances. On activation, these pathways result in theformation of protease complexes, the C3-convertases. The classicalpathway C3-convertase, C4b2a, and the alternative pathway C3-convertase,C3bBb, are both able to cleave the a chain of C3 generating C3b. C3b hasthe potential to bind covalently to biological surfaces. C3b bindingleads to opsonization for phagocytosis by polymorphonuclear cells andmacrophages. When additional C3b is available, the C3-convertases canfunction as C5-convertases, cleaving C5 and initiating the assembly ofthe TCC, or membrane attack complex (MAC), which mediates cellular lysisby insertion of pore-forming protein complexes into targeted cellmembranes.

In the classical pathway as shown in FIG. 1A, C1q, a collagenoussubcomponent of the first component (C1), binds to immunoglobulinswithin immune complexes, and its associated serine proteases, C1r andC1s, become activated. This complement cascade is initiated by thesubsequent cleavage of C4 and C2, followed by C3 activation. Theresulting C3b fragment not only acts as an opsonin but also leads to themembrane attack complex (MAC) formation in the lytic pathway. In innateimmunity, a complex composed of a recognition molecule (lectin) andserine proteases, termed the mannose-binding lectin (MBL)-associatedserine protease (MASP), activates C4 and C2 upon binding tocarbohydrates on the surface of microorganisms via the lectin pathway.This binding occurs in the absence of immunoglobulins. Recognitionmolecules of the lectin pathway found in jawed vertebrates are MBLs andficolins, both of which are characterized by the presence of acollagen-like domain, like C1q, and a carbohydrate binding domain havinga common binding specificity for GlcNAc. MASPs and C1r/C1s share thesame domain organization and form a subfamily of serine proteases.

The lectin complement pathway in innate immunity is closely related tothe classical complement pathway in adaptive immunity, e.g., withrespect to the structures and functions of their components. Bothpathways are typically initiated by complexes consisting of collagenousproteins and serine proteases of the mannose-binding lectin(MBL)-associated serine protease (MASP)/C1r/C1s family. It has beenspeculated that the classical pathway emerged evolutionarily after thelectin pathway.

Activation of the alternative complement pathway, shown in FIG. 1B,typically begins when C3b protein (or C3i) binds to a cell and othersurface components, e.g., of microbes. C3b can also bind toimmunoglobulin G (IgG) antibodies. Alternative pathway Factor B proteinthen combines with the C3b protein to form C3bB. Factor D protein thensplits the bound Factor B protein into fragments Bb and Ba, formingC3bBb. Properdin then binds to the Bb to form C3bBbP that functions as aC3 convertase capable of enzymatically splitting typically hundreds ofmolecules of C3 into C3a and C3b. Some of the C3b subsequently binds tosome of the C3bBb to form C3bBbC3b, a C5 convertase capable of splittingmolecules of C5 into C5a and C5b.

Since C3b is free in the plasma, it can bind to either a host cell orpathogen surface. To prevent complement activation from proceeding onthe host cell, there are several different kinds of regulatory proteinsthat disrupt the complement activation process. Complement Receptor 1(CR1 or CD35) and DAF (also known as CD55) compete with Factor B inbinding with C3b on the cell surface and can even remove Bb from analready formed C3bBb complex. The formation of a C3 convertase can alsobe prevented when a plasma protease called Factor I cleaves C3b into itsinactive form, iC3b. Factor I works with C3b-binding protein cofactorssuch as CR1 and Membrane Cofactor of Proteolysis (MCP or CD46). Anothercomplement regulatory protein is Factor H which either competes withfactor B, displaces Bb from the convertase, acts as a cofactor forFactor I, or preferentially binds to C3b bound to vertebrate cells.

The precise function of the complement system depends on its regulation,as activation of the complement cascade leads to the production of anumber of proteins that contribute to inflammation. This is beneficialwhen contributing to a host defense, but can be detrimental if activatedon self tissue. Typically, activation of C3 in the blood is kept at alow level, and C3b deposition is limited to the surface of pathogens.

The human wild type complement factor B protein is a 764 amino acid,single-chain glycoprotein (approximately 93-kDa) composed of fiveprotein domains (Mole et al., 1984 The J. Biol Chem, 259:6, 3407-3412).A human wild type complement factor B protein (fB) is typicallyexpressed with an N-terminal 25 amino acid signal peptide, e.g., see SEQID NO:1. The amino-terminal region (Ba) of human wild type complementfactor B protein consists primarily of three short consensus repeats.The middle region is a type A domain similar to those found in vonWillebrand factor (Colombatti et al., Blood (1991) 77(11):2305-15). Thecarboxy terminus is a serine protease (SP) domain (Perkins and Smith,Biochem J. (1993) 295(Pt 1):109-14; Hourcade et al., JBC (1998)273(40):25996-6000; Hourcade et al. J Immunol. (1999) 162(5):2906-11; Xuet al., J Biol Chem. 2000 275(1):378-85; Milder et al. Nat Struct MolBiol (2007) 14(3):224-8).

Complement factor B analogs and their use for inhibiting complement andtreating complement mediated diseases are described in PCT PublicationNo. WO08/106644 and U.S. Patent Publication No. US20100120665. Forexample, the human complement factor B protein analog, hfB3 (describedin U.S. Patent Publication No. 20100120665), is a dominant negativehuman factor B protein variant that efficiently inhibits the alternativecomplement (AP) activity. hfB3 protein (SEQ ID NO:4) has five amino acidchanges compared to a human wild type factor B protein (SEQ ID NO:1).The five amino acid changes enable hfB3 protein to (i) bind much tighterto C3b protein, (ii) resist C3b-dependent cleavage by factor D protein,and (iii) bind tighter to factor D protein when compared to the wildtype factor B protein. The tighter binding of hfB3 protein with C3bprotein and factor D protein sequester two essential components of thealternative complement pathway (ACP) in an inactive C3 convertase(hfB3), blocking the AP activity. Since C3b-bound hfB3 protein cannot becleaved by factor D protein, the conformational change of hfB3 proteindoes not occur and the serine protease at the C-terminus of hfB3 proteinis not activated.

Both human wild type complement factor B protein and hfB3 contain 23cysteine amino acids. The “active” forms of both have all of thecysteines forming disulfide bonds with one of the other cysteines, withthe exception of the cysteine corresponding to the C292 of SEQ ID NO:1.The C292 of the “active forms” of hfB3 and wild type factor B is a freecysteine (Parkes et al. 1983 Biochem J. 213, 201-209) and is highlyconserved among various mammalian species, e.g., see Table 1, below.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

SUMMARY OF THE INVENTION

The invention provides polypeptides comprising a complement factor Banalog. The invention also provides various complement factor B analogs.In some embodiments, a complement factor B analog comprises a mutationof a free cysteine amino acid. The invention also provides nucleic acidsand viral vectors comprising a nucleotide sequence encoding polypeptidesand complement factor B protein analogs of the invention. Someembodiments of the invention provide cells, wherein the cells comprise anucleic acid encoding a complement factor B protein analog of theinvention and wherein the cells express a complement factor B proteinanalog.

Additionally, the invention provides pharmaceutical preparationscomprising a polypeptide or complement factor B protein analog of theinvention, a nucleic acid of the invention, a viral vector of theinvention or any combination thereof.

Also provided by the present invention are methods of treating acomplement-mediated disease comprising administering to a patient apharmaceutical preparation of the invention, a polypeptide of theinvention, a complement factor B protein analog of the invention, anucleic acid of the invention, a viral vector of the invention or anycombination thereof.

The invention also provides methods of producing a polypeptidecomprising a complement factor B protein analog, the method comprising:expressing in a cell a complement factor B protein analog of theinvention and purifying said complement factor B protein analog.

Polypeptides of the invention, complement factor B analogs of theinvention, and nucleic acids and vectors encoding them, can be used tomodulate a complement pathway and for the study and/or treatment ofvarious conditions or diseases related to a complement pathway.

The invention is based on the findings that mutation or removal of afree cysteine (i) improves the yield of an active and/or properly foldedcomplement factor B protein analog (e.g., see Examples 9 and 10); (ii)enhances the thermostability of a complement factor B protein analog(e.g., see Examples 13 and 14); and/or (iii) reduces aggregation of acomplement factor B protein analog (e.g., see Example 6).

This summary of the invention does not necessarily describe all featuresor necessary features of the invention. The invention may also reside ina sub-combination of the described features.

BRIEF DESCRIPTION OF THE FIGURES

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities ofembodiments depicted in the drawings.

FIG. 1A depicts the classical and lectin complement pathways. Theclassical pathway is initiated through C1 while the lectin pathway isinitiated through mannose binding lectin (MBL). C4bC2a is a proteasethat cleaves C3 to C3a and C3b and is termed the C3 convertase.Similarly, C4bC2aC3b cleaves C5 to C5a and C5b and is termed the C5convertase. C3a, C4a, and C5a have inflammatory properties and attractphagocytotic cells. C5b6-9 forms the membrane attack complex (MAC),which creates membrane pores that kill infectious agents but can alsodamage host cells. MASP is mannan-binding lectin associated serineprotease.

FIG. 1B depicts the alternative complement pathway. This pathway isconstitutively active at a low level through spontaneous cleavage of C3.In the presence of an appropriate surface, C3b binds to complementfactor B (fB). This complex is then cleaved by complement factor D (fD)to yield C3bBb. Spontaneous dissociation (“decay”) of this complexwithin minutes leads to its inactivation, whereas stabilization byproperdin generates a complex that cleaves C3; that is, a C3 convertase.Several of the factors that attenuate the complement pathways do so byaccelerating the decay of the C3 and C5 convertases. C3b participates inthe C3 convertase to generate additional C3b thereby creating a positivefeedback loop as shown by the large arrow. C3bBb is a C3 convertase.C3bBbC3b is a C5 convertase.

FIG. 2 is a hfB3-292S expression construct. CMV-cytomegalovirusimmediate early promoter; IRES-internal ribosomal entry site;Neo-neomycin phosphotransferase gene; SynPolyA-synthetic polyA;Amp-Ampicillin-resistant gene. SEQ ID NO:8 is the nucleotide sequence ofthe hfB3-2925 expression construct shown in FIG. 2.

FIG. 3 shows Western blot analysis of raw cell culture supernatantscontaining either hfB3 or hfB3-2925 protein after cell cultureincubation for 72 hour (2×10⁶ cells/mL). Lane 1, one μl of cell culturemedium from naïve un-transfected 293 FreeStyle cells served as anegative control; Lane 2, one hundred ng of human wild type factor B(Quidel, Santa Clara, Calif.) purified from plasma served as a positivecontrol; Lane 3, one μl of cell culture medium from hfB3 producingcells; Lane 4, one μl of cell culture medium from hfB3-2925 producingcells. The molecular weight markers in KDa are indicated on the right.

FIG. 4 shows the results of a hemolytic assay. These results demonstrateinhibition of human alternative complement pathway hemolytic activity byraw hfB3 protein or hfB3-2925 protein producing cell culture medium. Therelative hemolytic activity is scored by hemoglobin released afterhemolysis of rRBCs by human alternative complement pathway activity.X-axis from left to right: factor B-depleted human serum supplementedwith 0 μg of purified human factor B protein (control, no lysis ofrRBCs); factor B-depleted human serum supplemented in each reaction witha mixture of 0.5 μg of purified human factor B protein and 1.0, 0.5,0.25 or 0.125 μg of hfB3 protein or hfB3-2925 protein, as indicated,from the culture medium of hfB3 protein or hfB3-2925 protein producingcells. Note: 100% inhibition represented no lysis of rRBCs. The Y-axisrepresents mean OD405 and Standard Deviation (SD).

FIG. 5A shows hfB3 protein (200 ng), purified from a three stepchromatography process, subjected to SDS-PAGE and silver staininganalysis. FIG. 5B shows inhibition of human alternative complementpathway hemolytic activity by the purified hfB3 protein. X-axis fromleft to right: factor B protein-depleted human serum supplemented with 0μg of purified wild type human factor B protein (wt hfB) (control, nolysis of rRBCs); factor B protein-depleted human serum supplemented ineach reaction with a mixture of 0.5 μg of purified wild type humanfactor B protein and 1.0, 0.5, 0.3, 0.2, 0.1 or 0.05 μg of hfB3, asindicated. 100% inhibition represents no lysis of rRBCs. The Y-axisrepresents mean OD405 and SD.

FIG. 6 shows biological activity of two populations of hfB3 protein.Shown are the results for hydrophobic interaction chromatography (HIC)purified hfB3 protein from Peak I and Peak II for inhibition of humanalternative complement pathway hemolytic activity. X-axis from left toright: factor B protein-depleted human serum supplemented with 0 μg ofpurified human factor B protein (control, no rRBCs lysis); factor Bprotein-depleted human serum supplemented in each reaction with amixture of 0.5 μg of purified wild type human factor B protein andvarious amounts of hfB3 protein ranging from 1.0 to 0.05 μg. 100%inhibition represents no lysis of rRBCs. The Y-axis represents meanOD405 and SD.

FIG. 7A shows reverse phase high-pressure liquid chromatography (HPLC)of raw cell culture supernatants containing either hfB3 protein orhfB3-2925 protein after tissue culture incubation for 72 hours (2×10⁶cells/mL). Supernatants from hfB3 producing cells ( . . . ), hfB3-2925protein producing cells (______) and naïve 293 cells (_ _ _ _ _), wereapplied to an Agilent HP1100 HPLC system using a narrow bore Jupiter™ C4column (Phenomenex) and eluted with a 50 minute water/acetonitrile(25-70% acetonitrile) gradient containing 0.1% TFA. Elution wasmonitored at 215 nm with a PDA detector. The position of heat shock 70protein (HSP70), present in all three samples, is also shown.

FIG. 7B shows an enlarged region of the chromatogram shown in FIG. 7Afocusing on the region (25-29 minutes) containing the Peaks I and II ofhfB3 protein and the peak containing hfB3-2925 protein.

FIG. 8 shows analysis of hfB3-Fc protein expression by subjecting 2 μlof cell culture supernatant containing hfB3-Fc protein to a non-reducingSDS-PAGE and Western blot analysis. (See Example 12) Two bands ofhfB3-Fc protein were detected with molecular weight markers in KDaindicated on the left.

FIG. 9 shows representative H&E staining of paraffin sections of theright front paw joints of mice from a study testing hfB3-2925 in acollagen antibody-induced arthritis (CAIA) mouse model for rheumatoidarthritis as described in Example 16. Group 1 is the vehicle controlgroup that received no collagen antibody cocktail. Group 2 is theuntreated group that received the collagen antibody cocktail. Group 3 isthe treated group that received the collagen antibody cocktail and wastreated with hfB3-2925.

FIG. 10 shows joint swelling measurements from a study testing hfB3-2925in a collagen antibody-induced arthritis (CAIA) mouse model forrheumatoid arthritis as described in Example 16. The groups are the sameas those in FIG. 8, as described in the paragraph. hfB3-292S caused astatistically significant (p<0.0003) reduction in joint swelling ascompared to the untreated group (Group 2).

FIG. 11 shows a Western blot analysis of cell culture medium from cellstransfected with an hfB3-292SN480 expression construct. This Westernblot analysis was performed using a monoclonal antibody specific forhfB3-292S. The left lane contains hfB3-292S and the right lane is cellculture medium from cells transfected with an hfB3-292SN480 expressionconstruct. The analysis detected a band of approximately 55 KDa from thecell culture medium of hfB3-292SN480 cell line (right lane).

FIG. 12 shows a Western blot analysis of cell culture medium from cellstransfected with an hfB3-292S/Fc-mono expression construct as describedin Example 19, below. A band of approximately 115 KDa was detected bypurified goat anti-human factor B antibody from this non-reducingSDS-PAGE.

FIG. 13 shows the cell culture supernatant from cells expressinghfB3-292SN480 inhibited the human alternative complement pathwayhemolytic activity in a dose dependent manner.

FIG. 14 shows the cell culture supernatant from cells expressinghfB3-292S/Fc-mono inhibited the human alternative complement pathwayhemolytic activity in a dose-dependent manner.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1—amino acid sequence of a wild-type human complement factor B

SEQ ID NO:2—amino acid sequence of a human complement factor B proteinanalog, hfB3-2925, which comprises the following mutations: K258A,R259A, K260A, D279G, N285D and C292S.

SEQ ID NO:3—amino acid sequence of a human complement factor B analog,hfB3-2925-740N, comprising the following mutations as compared to SEQ IDNO:1: K258A, R259A, K260A, D279G, N285D, D740N and C292S.

SEQ ID NO:4—amino acid sequence of a human complement factor B analog,hfB3, which comprises the following mutations as compared to SEQ IDNO:1: K258A, R259A, K260A, D279G and N285D.

SEQ ID NO:5—nucleotide sequence of an hfB3 expression construct, theconstruction of which is described in Example 1.

SEQ ID NO:6-7—primers for site directed mutagenesis SEQ IDNO:8—nucleotide sequence of an hfB3-2925 expression construct, theconstruction of which is described in Example 2.

SEQ ID NO:9-14—partial amino acid sequences of complement factor Bproteins from a human, a mouse, a rat, a pig, a monkey and a sheep,respectively.

SEQ ID NO:15-16—primers for site directed mutagenesis SEQ ID NO:17—aminoacid sequence of a human complement factor B protein analog, hfB4.

SEQ ID NO:18—nucleotide sequence of an hfB3-Fc expression construct, theconstruction of which is described in Example 12.

SEQ ID NO:19-20—primers for site directed mutagenesis.

SEQ ID NO:21—amino acid sequence of a human complement factor B proteinanalog, hfB3-Fc.

SEQ ID NO:22—amino acid sequence of a human complement factor B proteinanalog, hfB3-2925-Fc.

SEQ ID NO:23—amino acid sequence of a human complement factor B proteinanalog, hfB3-2925-740N-Fc.

SEQ ID NO:24—nucleotide sequence of an expression construct forexpressing hfB3-292SN480.

SEQ ID NO:25—nucleotide sequence gene expression construct forhfB3-2925/Fc-mono.

SEQ ID NO:26—amino acid sequence of hfB3-2925/Fc-mono.

SEQ ID NO:27—amino acid sequence of an Fc domain.

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, molecular biology,cell culture, virology and the like which are in the skill of one in theart. These techniques are fully disclosed herein and/or in currentliterature, for example, Sambrook, Fritsch and Maniatis eds., “MolecularCloning, A Laboratory Manual”, 2nd Ed., Cold Spring Harbor LaboratoryPress (1989); Celis J. E. “Cell Biology, A Laboratory Handbook” AcademicPress, Inc. (1994) and Bahnson et al., J. of Virol. Methods, 54:131-143(1995).

It is contemplated that any method, preparation or composition describedherein can be implemented with respect to any other method, preparationor composition described herein. The use of the word “a” or “an” whenused in conjunction with the term “comprising” in the claims and/or thespecification may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.” The use ofthe term/phrase “and/or” when used with a list means one or more of thelisted items may be utilized, e.g., it is not limited to one or all ofthe elements.

During production and purification of the complement factor B proteinanalog designated hfB3 (SEQ ID NO:4), two populations of the complementfactor B protein analog were detected. One population had the desiredactivity for the complement factor B protein analog (Peak I) while theother population had substantially less of the desired activity (PeakII), e.g., see FIGS. 6, 7A and 7B. The results from characterization ofthe two populations suggested that the two populations differed in theirdisulfide bond patterns. When the free cysteine (position 292 of SEQ IDNO:4) was mutated to a serine, Peak II was undetectable. FIG. 6 showsthat the Peak II fraction exhibits some ability to inhibitcomplement/hemolytic activity, but much less ability per ug of proteinas compared to the Peak I fraction. It is possible that thecomplement/hemolytic inhibitory activity seen with Peak II is mostly orsolely a result of Peak II being containing some hfB3 with a freecysteine at position 292, possibly as a result of Peak I and Peak II notbeing fully resolved from each other.

The cysteine corresponding to position 292 of SEQ ID NO:1 is highlyconserved among complement factor B proteins of different mammalianspecies (e.g., see Table 1). Highly conserved sequences are typicallyimportant to the function of a protein. “The neutral theory of molecularevolution states that mutations in amino acids occur in a stochasticallyconstant manner as long as the mutations have no effect on the functionof the gene product [Kimura M: The neutral theory of molecularevolution. Sci Am 1979, 241(5):98-100, 102, 108 passim]. On the otherhand, amino acids that are important for protein function and structurecannot mutate without a detrimental effect on protein activity.Therefore, these amino acids will change very slowly in a given proteinfamily during evolution.” (Liu et al. BMC Bioinformatics 2006, 7:37)

TABLE 1 Conserved Cysteine in Complement Factor B Protein HUMAN*IGASNFTGAKKCLVNLIEKVASY (SEQ ID NO: 9) MOUSE IGSSNFTGAKRCLTNLIEKVASY(SEQ ID NO: 10) RAT IGASNFTGAKRCLANLIEKVASY (SEQ ID NO: 11) PIGIGARNFTGAKNCLKDFIEKVASY (SEQ ID NO: 12) MONKEY IGAGNFTGAKKCLVNLIEKVASY(SEQ ID NO: 13) SHEEP VGAHNFTGAKNCLRDFIEKVASY (SEQ ID NO: 14) *Ccorresponds to position 292 for a human factor B (SEQ ID NO: 1)

Mutating the cysteine at amino acid 292 of the complement factor Bprotein analog to a serine, e.g., as shown in SEQ ID NO:2 (hfB3-292),greatly reduced, if not eliminated, the amount of the Peak II(substantially less active) population and the hfB3-2925 complementfactor B protein analog retained its activity, in this case the abilityto inhibit or reduce complement activity. (E.g., see Example 10, below.)

Not wishing to be bound by theory, the less active population (Peak IIfraction) of hfB3 protein could be the result of misfolding of hfB3protein. It is possible that the generation of the majority of Peak IIpopulation was due to the combination of the free cysteine and themutations introduced to hfB3 protein because when cells were engineeredto express a wild-type human complement factor B protein (SEQ ID NO:1)in a manner similar to that used for hfB3 protein, only one populationof the wild-type human factor B protein was detected (data not shown).

This mutation of a free cysteine allows for a higher yield of the activecomplement factor B analog since most, if not all, of the producedcomplement factor B protein analog is in an active form. Additionally,mutation of a free cysteine results in a more stable protein since allof the remaining unmutated cysteines are part of a disulfide bond andthere is no free cysteine left that can participate in possiblyundesirable and detrimental reactions. Additionally, mutating the freecysteine unexpectedly appears to have reduced or eliminated aggregationof the complement factor B protein analog, e.g., see Example 6 and FIG.3.

The complement factor B protein analogs described in PCT Publication No.

WO08/106644 or U.S. Patent Publication No. US20100120665 (both of whichare incorporated by reference in their entirety) can have their freecysteine mutated and still retain their desired function, whilebenefiting from the aforementioned advantages of the mutation.Therefore, the present invention includes any of the complement factor Bprotein analogs described in PCT Publication No. WO08/106644 or U.S.Patent Publication No. US20100120665 with a mutation of a free cysteine.

The term “free cysteine” refers to a cysteine that is part of a proteinor a peptide, wherein the free cysteine does not form a disulfide bondwith another cysteine in the same protein or peptide. In some cases a“free cysteine” of a protein analog does not form a disulfide bond withanother cysteine (in the same protein or peptide) when the proteinanalog has a desired activity, but may form a disulfide bond withanother cysteine (in the same protein or peptide) in a less active orinactive form of the protein analog.

The term “complement-mediated” refers to a process or disease thatinvolves complement. Typically, a “complement-mediated” disease orcondition is one wherein complement activity is one of the underlyingcauses of the disease or condition and wherein inhibition or blocking ofthe complement activity lessens the extent of the disease or condition.Examples of numerous complement-mediated diseases or conditions aredescribed herein.

The term “wildtype” (or wild-type), which is used interchangeably with“native”, relates to a naturally occurring protein encoded by amammalian genome, a naturally occurring nucleic acid, and so on. In somecases, there may be actually more than one protein corresponding to thewild-type version, e.g., due to allelic differences; different isoforms;and/or genetic variation among different individuals of a species.

The term “analog” refers to a structural derivative of a protein (parentprotein). An analog does not necessarily retain all of the properties ofthe parent protein and in some cases has at least one altered propertyas compared to the corresponding native parent protein. In someembodiments, a parent protein is a native (naturally-occurring) protein.An analog or variant protein is produced by replacing, substituting,deleting, and/or adding amino acids with regard to the correspondingnative amino acid sequence of the protein. The substitutions orinsertions typically involve naturally occurring amino acids, but mayalso include synthetic or unconventional amino acids as well. In someembodiments, an analog or variant is produced by mutating a protein,e.g., mutating a nucleic acid encoding it. An analog will typicallyretain at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, at least 99.5% of the corresponding native parent protein's aminoacid sequence (e.g., have that percent amino acid sequence identity withrespect to the naturally occurring parent protein as determined over thelength of the entire parent protein or, in certain embodiments, over aspecific domain or portion of the parent protein). Analogs also includefragments of full length analogs that comprise a portion of the aminoacid sequence and either retain one or more biological activities of theparent protein or of a full length analog or inhibit one or more ofthese biological activities.

The term “corresponds” or “corresponding” when referring to an aminoacid in a particular protein refers to the particular amino acid in thatparticular protein and also to an amino acid in a related or similarprotein and may provide a similar function to the protein. For example,an amino acid in a human complement factor B may be found to correspondwith an amino acid in a murine complement factor B or in a human allelicvariant of factor B, usually determined by aligning the two amino acidsequences. For example, one skilled in the art can align two or morerelated sequences, such as SEQ ID NOs:9-14, to determine correspondingamino acids, e.g., using a BLAST program (e.g., see Table 1, above).Also, corresponding amino acids can be determined, e.g., by aligningmotifs (e.g., a protease cleavage motif) within related or unrelatedproteins. Such an alignment may also be used to derive consensussequences of target protein or domains thereof.

As used herein, the term “gene” typically refers to a coding region fora protein. However, in some contexts herein it will be clear that theterm “gene” is also referring to elements (e.g., regulatory elements)operatively linked to a coding region such as promoters, enhancers,splice sites (acceptors and/or donors), polyadenylation signals,introns, 5′ untranslated regions, 3′ untranslated regions, etc.

The term “pharmaceutically acceptable” means approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inhumans.

A “therapeutic benefit” is not necessarily a cure for a particulardisease or condition (including any disease or condition describedherein), but rather, encompasses a result which most typically includesalleviation of the disease or condition, elimination of the disease orcondition, reduction of one or more symptoms associated with the diseaseor condition, prevention or alleviation of a secondary disease orcondition resulting from the occurrence of a primary disease orcondition, diminishing the likelihood of developing a condition ordisease, diminishing the severity of a disease or condition, changingthe character of a disease or condition, shortening the course of adisease or condition, slowing or preventing the progression or worseningof a disease or condition, and/or prevention of the disease orcondition.

Complement Factor B Analogs

The present invention includes complement factor B protein analogs andpolypeptides comprising complement factor analogs and their uses. Someembodiments of the invention include a complement factor B proteinanalog wherein a free cysteine has been mutated. In some embodiments,this mutation of a free cysteine can comprise a deletion of the freecysteine or substitution of the free cysteine with another aminoacid(s). A free cysteine can be substituted with essentially any aminoacid, that still allows for the complement factor B protein analog toretain at least some of the desired characteristic(s), such as theability to downregulate, diminish or ablate complement activity. Asubstitution may be with one or more amino acids. In some embodiments, afree cysteine is substituted with a serine. In some embodiments, a freecysteine is substituted with one or more amino acids selected from thegroup consisting of alanine, histidine, isoleucine, leucine, methionine,phenylalanine, serine, threonine, tyrosine and valine. In someembodiments, a free cysteine corresponds to amino acid 292 of SEQ IDNO:1.

In some embodiments, the invention provides complement factor B proteinanalogs that do not comprise a free cysteine. The invention alsoprovides methods of making or producing a complement factor B proteinanalog comprising mutating a free cysteine.

Mutation of a free cysteine can be combined with other mutations of acomplement factor B protein, e.g., other mutations as described herein.

The invention also provides complement factor B protein analogs whereinthe cysteine corresponding amino acid 292 of SEQ ID NO:1 is mutated.This mutation can be a deletion, insertion or substitution, such as aserine substitution or other mutations as described herein.

Analogs can include various muteins of a sequence other than thenaturally-occurring amino acid sequence. For example, single or multipleamino acid substitutions (e.g., conservative or non-conservative aminoacid substitutions) may be made in the naturally-occurring sequence. Aconservative amino acid substitution generally should not substantiallychange the structural characteristics of the parent sequence (e.g., areplacement amino acid should not tend to break a helix that occurs inthe parent sequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al. Nature 354:105 (1991). Conservative substitutionsinclude, but are not limited to, those from the following groupings:Acidic Residues Asp (D) and Glu (E); Basic Residues Lys (K), Arg (R),and His (H); Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn (N),and Gln (Q); Aliphatic Uncharged Residues Gly (G), Ala (A), Val (V), Leu(L), and Ile (I); Non-polar Uncharged Residues Cys (C), Met (M), and Pro(P); Aromatic Residues Phe (F), Tyr (Y), and Trp (W); Alcoholgroup-containing residues S and T; Aliphatic residues I, L, V and M;Cycloalkenyl-associated residues F, H, W and Y; Hydrophobic residues A,C, F, G, H, I, L, M, R, T, V, W and Y; Negatively charged residues D andE; Polar residues C, D, E, H, K, N, Q, R, S and T; Positively chargedresidues H, K and R; Small residues A, C, D, G, N, P, S, T and V; Verysmall residues A, G and S; Residues involved in turn formation A, C, D,E, G, H, K, N, Q, R, S, P and T; and Flexible residues Q, T, K, S, G, P,D, E and R.

In some embodiments, a non-conservative substitution is used.

In some embodiments, mutations include, but are not limited to,substitutions of one or more amino acids, deletions of one or more aminoacids or insertions of one or more amino acids. Mutations include, butare not limited to, those which: (1) reduce susceptibility of thecomplement factor B analog to proteolysis, (2) reduce susceptibility ofthe complement factor B analog to oxidation, (3) alter binding affinityof the complement factor B analog for forming protein complexes, (4)alter (e.g., increase or decrease) binding affinities of the complementfactor B analog, (5) reduce immunogenicity of the complement factor Banalog; (6) increase stability (e.g., thermostability) of the complementfactor B analog; (7) reduce aggregation of the complement factor Bprotein analog; or any combinations of 1-7.

In some embodiments, a human complement factor B analog of the inventioncompetes with binding of native complement factor B. For example nativecomplement factor B can bind with complement factor C3b to form C3bB,e.g., see FIG. 1B. Factor B that is part of the C3bB complex can bindfactor D. Therefore, in some embodiments, a complement factor B analogof the invention can compete with the binding of a native factor B for(i) binding to C3b, (ii) binding to factor D or (iii) both.

In some embodiments, a complement factor B protein analog of theinvention, is an analog of SEQ ID NO:4 having cysteine amino acids thatform disulfide bonds and a free cysteine amino acid that has beensubstituted by another amino acid, more than one amino acid or has beendeleted with no substitution.

In some embodiments of the invention, a complement factor B proteinanalog has increased C3b binding affinity as compared to a correspondingnative complement factor B protein and the complement factor B proteinanalog comprises (i) diminished protease activity as compared to acorresponding native complement factor B protein; (ii) diminishedability to be cleaved by factor D protein as compared to a correspondingnative complement factor B protein; or (iii) diminished proteaseactivity as compared to a corresponding native complement factor Bprotein and diminished ability to be cleaved by a factor D protein ascompared to a corresponding native complement factor B protein.

In some embodiments, a complement factor B analog comprises a mutationin the C3b binding domain and the complement factor B protein analogexhibits increased binding affinity to C3b as compared to the bindingaffinity of a corresponding native complement factor B protein to C3b.In some embodiments, a mutation in the C3b binding domain comprises (i)a substitution or deletion of an aspartic acid corresponding to aminoacid 279 of SEQ ID NO:1, a substitution or deletion of an asparaginecorresponding to amino acid 285 of SEQ ID NO:1 or both; or (ii) aninsertion of at least one amino acid next to said aspartic acid or saidasparagine. In some embodiments, this aspartic acid, asparagine or bothare substituted with one or more amino acids. In some embodiments, anaspartic acid corresponding to amino acid 279 of SEQ ID NO:1 issubstituted with glycine, alanine or asparagine. In some embodiments, anasparagine corresponding to amino acid 285 of SEQ ID NO:1 is substitutedwith glycine, alanine, or aspartic acid. In some embodiments, anaspartic acid corresponding to amino acid 279 of SEQ ID NO:1 issubstituted with glycine and an asparagine corresponding to amino acid285 of SEQ ID NO:1 is substituted with aspartic acid.

In some embodiments, a complement factor B protein analog is a humancomplement factor B protein analog, based on a human complement factorprotein.

The instant invention includes complement factor B protein analogs,e.g., that can be delivered as proteins and/or via gene transfer toattenuate the alternative pathway of complement activation. Theseanalogs may overcome hurdles that impede the development of somecomplement inhibitors including, for example: 1) avoiding long termsystemic immune suppression; 2) achieving efficacy in the face ofotherwise prohibitively high levels of complement factors in the blood;3) achieving sufficient levels and distribution of the therapeuticcomplement factor B protein analog in the proximity of the retina andBruch's membrane for efficacy; 4) achieving activity of the therapeuticcomplement factor B protein analog within drusen; 5) achievingsufficient duration of therapeutic delivery to treat a chronic disease;6) achieving efficacy without detrimentally interfering with theclassical complement pathway activities in the back of the eye; and/or7) avoiding or diminishing an immune reaction (e.g., a local immunereaction) to the therapeutic.

Attenuating the positive feedback loop in the alternative pathway is ameans of down-regulating the entire alternative pathway. One suitablemeans for attenuating the alternative pathway feedback loop is tointerfere with complement factor B (fB) protein function or levels. Someembodiments of the invention use a complement factor B analog forattenuating complement activity.

A complement factor B protein analog of the invention may comprise atleast one mutation corresponding to a mutation of SEQ ID NO:1 selectedfrom the group consisting of K258A, R259A, K260A, D279G, N285D andD740N. In some embodiments, it comprises mutations corresponding toK258A, R259A, K260A, D279G and N285D of SEQ ID NO:1. In someembodiments, a complement factor B protein analog comprises a mutationcorresponding to D740N of SEQ ID NO:1.

For exemplary purposes, specific analogs of complement factor B proteinare described herein. Factor B protein can be manipulated in a number ofways, e.g., to inhibit or reduce activation of the alternative pathway.In some embodiments, particular sites in factor B can be altered, forexample, by site directed mutagenesis, so that the molecule no longerfully functions properly. In some embodiments, the enzyme portion ordomain, (e.g., the protease domain, which is a serine protease) of themolecule can be altered so that the molecule no longer has enzymaticactivity or has reduced enzymatic activity (e.g., reduced by at least 2fold, 5 fold, 10 fold, 50 fold or 100 fold). In some embodiments, acomplement factor B protein analog comprises a mutation in the activesite of the serine protease domain, wherein the mutation decreases orablates the complement factor B protein analog's ability to cleavecomplement factor C3 as compared to a corresponding native complementfactor B protein.

In some embodiments, this can be achieved by altering the residuecorresponding to amino acid 740 of SEQ ID NO:1. In some embodiments,this mutation comprises a deletion or a substitution of an aspartic acidcorresponding to amino acid 740 of SEQ ID NO:1. In some embodiments, anaspartic acid (D), corresponding to amino acid 740 of SEQ ID NO:1, issubstituted with another amino acid such as asparagine (N), alanine (A),glutamic acid (E), serine (S), tyrosine(Y), or glycine (G). Thenumbering of particular factor B amino acids herein relates to theentire polypeptide including the signal peptide and is reflected in SEQID NO:1. Hourcade et al. (JBC (1998) 273(40):25996-6000) notes thatamino acids 739-746 of SEQ ID NO:1 (referred to in Hourcade et al. asamino acids 714-721 because the Hourcade et al. numbering does notinclude the 25 amino acid signal sequence/peptide) play a role in theserine protease function of factor B protein. Additionally, N693, T694and D740 may constitute or be part of the substrate binding site andH526, D576 and 5699 may constitute or be part of the catalytic center,e.g., see Xu et al., J Biol Chem. 2000 275(1):378-85. In someembodiments, a factor B protein analog comprises a mutation of at leastone of the amino acids selected from amino acids 739-746 of SEQ ID NO:1.In some embodiments, a complement factor B protein analog comprises asubstitution of the amino acid corresponding to amino acid 739 of SEQ IDNO:1 with an alanine. In some embodiments, a complement factor B proteinanalog comprises a substitution of the amino acid corresponding to aminoacid 740 of SEQ ID NO:1 with an amino acid selected from the groupconsisting of asparagine, glutamic acid, alanine, serine and tyrosine.In some embodiments, a complement factor B protein analog comprises asubstitution of the amino acid corresponding to amino acid 741 of SEQ IDNO:1 with an amino acid selected from the group consisting of tryptophanand alanine. In some embodiments, a complement factor B protein analogcomprises a substitution of the amino acid corresponding to amino acid742 of SEQ ID NO:1 with glutamine. In some embodiments, a complementfactor B protein analog comprises a substitution of the amino acidcorresponding to amino acid 743 of SEQ ID NO:1 with phenylalanine. Insome embodiments, a complement factor B protein analog comprises asubstitution of the amino acid corresponding to amino acid 745 of SEQ IDNO:1 with phenylalanine. In some embodiments, a complement factor Bprotein analog comprises a substitution of the amino acid correspondingto amino acid 746 of SEQ ID NO:1 with tryptophan or alanine. In someembodiments, a factor B protein analog comprises a mutation of one ortwo of the amino acids 693 and 694 of SEQ ID NO:1, e.g., a substitutionor deletion. In some embodiments, a factor B protein analog comprises amutation of one or two of the amino acids 526, 576 and 699 of SEQ IDNO:1, e.g., a substitution or deletion.

Other sites in factor B that can be altered include: 1) the binding sitefor properdin (the properdin binding domain) such that binding occurswith lower affinity (for example, such as 2 fold, 5 fold, 10 fold, 50fold or 100 fold reduced affinity as compared to the wild type factor Bprotein) or with greater affinity (such as at least 2 fold, 5 fold, 10fold, 50 fold or 100 fold increased affinity as compared to the wildtype factor B); 2) the binding site for C3b protein (the C3b bindingdomain) such that binding occurs with lower affinity (such as at least 2fold, 5 fold, 10 fold, 50 fold or 100 fold reduced affinity as comparedto the wild type factor B protein) or with greater affinity (such as atleast 2 fold, 5 fold, 10 fold, 50 fold or 100 fold increased affinity ascompared to wild type factor B protein, for example, this may beachieved by substituting the amino acid corresponding to position 279and/or position 285 of SEQ ID NO:1 with other amino acids, for example,wherein the amino acid at the position corresponding to position 279 issubstituted with asparagine (N), alanine (A) or glycine (G) and/or theamino acid at the position corresponding to position 285 is substitutedwith aspartic acid (D) or alanine (A)); 3) the site acted on by factor Dsuch that factor D has reduced ability to cleave or no longer cleavesfactor B to form Bb (for example, at the factor D cleavage site, atleast one of the amino acids at the positions corresponding to position258, 259 or 260 of SEQ ID NO:1, for example, can be altered to alanine(A) or; a combination of any of 1, 2, and/or 3 above).

In some embodiments, a complement factor B protein analog comprises analteration in the complement factor D cleavage site wherein thealteration decreases or ablates cleavage of the complement factor Bprotein analog by factor D protein. In some embodiments, an alterationin the factor D cleavage site comprises (i) a substitution or deletionof an arginine corresponding to amino acid 259 of SEQ ID NO:1, asubstitution or deletion of one or both lysines corresponding to aminoacid 258 or 260 of SEQ ID NO:1 or a substitution or deletion of thearginine and both lysines; or (ii) an insertion next to the arginine,next to the one or both lysines, or next to the arginine and one or bothof the lysines. In some embodiments, a complement factor B proteinanalog has amino acids corresponding to amino acids 258-260 of SEQ IDNO:1 each replaced with alanine.

The invention includes (i) complement factor B protein analogs that bindto both factors C3b and D; (ii) complement factor B protein analogs withincreased binding (as compared to their native form) to both factors C3band D; (iii) complement factor B protein analogs with increased binding(as compared to their native form) to factor D protein; and (iv)complement factor B protein analogs with increased binding (as comparedto their native form) to C3bB complex. The invention also includesmethods of inhibiting a complement pathway using the complement factor Bprotein analogs of the invention, such as i-iv, above.

In some embodiments, increased binding is increased by about 1.5 toabout 10,000, about 10 to about 10,000, about 100 to about 10,000, about1,000 to about 10,000, about 1.5 to about 1,000, about 1.5 to about 100,about 1.5 to about 10, about 2 to about 5, about 2 to about 10, about 5to about 10, about 5 to about 20, about 10 to about 20, about 10 toabout 30, about 20 to about 30, about 30 to about 50, about 50 to about100, about 100 to about 500, about 500 to about 1,000, about 1,000 toabout 5,000, or about 5,000 to about 10,000 fold. In some embodiments,increased binding is increased by greater than 1.5, 2, 3, 4, 5, 10, 50,100, 500, 1000, 5000 or 10,000 fold. In some embodiments, increasedbinding can be measured by immunoprecipitation or using a Biacore (GEHealthcare, Piscataway, N.J.), e.g., as compared to the wild typeprotein. As an example for (i) above, binding could be measured byimmunoprecipitation of the protein with a binding molecule for C3bprotein and then detecting factor D protein in the immunoprecipitate,e.g., using an immunoassay such as an ELISA or Western, for example,with increased binding demonstrated as a band of increased intensity ina Western.

Some factor B protein analogs of the invention may have increasedbinding to C3b protein and/or factor D protein by a factor of 2 fold, 4fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold, 500 fold, 1000 foldas compared to binding of wild type factor B to C3b and/or factor D. Insome embodiments, increased binding can be measured byimmunoprecipitation or using a Biacore (GE Healthcare, Piscataway,N.J.).

Some modified factor B protein analogs of the invention comprise one ormore of the amino acid alterations discussed herein and additionallyhave one or more additional amino acid substitutions, insertions oralterations (e.g., at least or no more than 1, 2, 5, 8, 10, 15 20, 50,100, or 200 alterations), which analogs retain the increased binding toC3b and/or factor D or other biological activity of the factor B proteinanalogs discussed herein, which mediates inhibition of the complementpathway. Such analogs may have at least 99.9%, 99%, 98%, 95%, 90%, 85%,80%, 75% or 70% amino acid sequence identity with a wild type factor Bprotein, for example, the amino acid sequence of SEQ ID NO:1 and retainthe increased binding to C3b and/or factor D.

In the context of gene therapy and expression of complement factor Bprotein analogs, the alterations/mutations will be reflected at thelevel of the encoding nucleic acid. Thus, modifications also can be madeto the nucleic acid to enhance expression. For example, certain codonsmay be preferred by a particular host cell. Thus, recoding can beperformed where certain codons are preferred in, for example, aparticular mammalian expression system or cell.

Some embodiments of the invention are directed to polynucleotides andhost cells (or host multicellular organisms) useful in the production ofa complement factor B protein analog and directed to the analogs, e.g.,hfB3-2925 (SEQ ID NO:2), hfB3-2925-740N (SEQ ID NO:3), hfB3-2925-Fc (SEQID NO:22), hfB3-2925-740N-Fc (SEQ ID NO:23) or hfB3-2925/Fc-mono (SEQ IDNO:26). Methods of isolating and testing complement-mediated activity ofthese complement factor B protein analogs are also provided. Someaspects of the invention are directed to pharmaceuticalcompositions/preparations wherein a complement factor B protein analogis an active ingredients in a therapeutic and/or a prophylacticcontexts. Some embodiments of the invention are also directed to methodsof treating complement-mediated disorders using a therapeuticallyeffective amount of a complement factor B protein analog.

In some embodiments of the invention, a complement factor B analog maycomprise glycosylation patterns which are distinct from glycosylationpatterns on a naturally-occurring complement factor B, or may lackglycosylation altogether. Carbohydrates may be added to and/or removedfrom factor B analogs comprising glycosylation site sequences for N-and/or O-linked glycosylation in vitro, such as with a canine pancreaticmicrosome system (e.g., see Mueckler and Lodish (1986) Cell 44:629 andWalter, P. (1983) Meth. Enzymol. 96:84) or the like. A complement factorB analog of the invention may be produced comprising adding ordeleting/mutating an amino acid sequence corresponding to aglycosylation site, e.g., changing the glycosylation pattern/status of aprotein can change functional characteristics of a protein. For example,hfB2, hfB3 (SEQ ID NO:4), hfB3-2925 (SEQ ID NO:2), hfB3-2925-740N (SEQID NO:3), hfB3-2925-Fc (SEQ ID NO:22), hfB3-2925-740N-Fc (SEQ ID NO:23)and hfB3-2925/Fc-mono (SEQ ID NO:26) comprise an N285D substitution, ascompared to a wildtype factor B (SEQ ID NO:1), which results in theremoval of an N-glycosylation site. (Both the hfB2 and hfB3 complementfactor B protein analogs are described in detail in U.S. PatentPublication No. US20100120665.) The loss of the N-glycosylation sitealters the characteristics of the protein. The same effect may beachieved by producing the protein in a cell that has an alteredglycosylation pattern or that does not glycosylate this N285. Forexample, a factor B protein or analog may be produced in an E. coli cellthat does not glycosylate the N285. In some embodiments, a complementfactor B protein analog is produced in a cell (e.g., an E. coli) thatdoes not glycosylate an amino acid corresponding to a wildtype factor Bprotein that is typically glycosylated, e.g., corresponding to aminoacid N285 of SEQ ID NO:1.

As demonstrated in PCT Publication No. WO08/106644 and U.S. PatentPublication No. US20100120665, in some cases a native factor B proteinfrom one species may have activity in a complement reaction/pathway fromanother species. Therefore, the present invention also includescomplement factor B protein analogs from one species for inhibitingcomplement activity in another species.

In some embodiments, a complement factor B protein analog of theinvention is PEGylated, e.g., see Roberts et al., Advanced Drug DeliveryReviews 54(4):459-476 (2002); Veronese, Biomaterials 22(5):405-417(2001); Fee and Alstine, Chemical Engineering Science 61(3):924-939(2006); Kodera et al., Progress in Polymer Science 23(7):1233-1271(1998); Morar, Biopharm International 19(4):34 (2006); and Veronese andPasut, Drug Discovery Today 10(21):1451-1458 (2005). Polyethylene glycol(PEG) can be attached to a complement factor B protein analog of theinvention. In some embodiments, PEG is attached with or without amultifunctional linker either through site-specific conjugation of thePEG (e.g., to the N-terminus or to the C terminus of a complement factorB analog) or via epsilon amino groups present on lysine residues. Insome embodiments, linear or branched polymer derivatization that resultsin minimal loss of biological activity can be used. The degree ofconjugation can be closely monitored by SDS-PAGE and mass spectrometryto ensure proper conjugation of PEG molecules to a complement factor Banalog. In some embodiments, unreacted PEG can be separated from PEGconjugates by size-exclusion and/or by ion exchange chromatography.

In certain embodiments, the carboxy-terminus, the amino-terminus or bothof a complement factor B analog, are chemically modified. Amino-terminalmodifications such as acylation (e.g., acetylation) or alkylation (e.g.,methylation) and carboxy-terminal modifications such as amidation, aswell as other terminal modifications, including cyclization, may beincorporated into various embodiments of the invention. Certainamino-terminal and/or carboxy-terminal modifications and/or peptideextensions (such as fused to a heterologous polypeptide, such asalbumin, immunoglobulin or portion thereof, such as an immunoglobulin Fcdomain) to the core sequence can provide advantageous physical,chemical, biochemical, and pharmacological properties, such as: enhancedstability, increased potency and/or efficacy, resistance to serumproteases, desirable pharmacokinetic properties, and others.

In some embodiments, a complement factor B protein analog of theinvention comprises an immunoglobulin Fc domain, for example, a humanimmunoglobulin Fc domain. In some embodiments, an Fc domain isC-terminal to the corresponding complement factor B analog amino acidsequence. In some embodiments, an Fc domain comprises or consists ofamino acids 766-990 of SEQ ID NO:21 or amino acids 766-1003 or 786-1003of SEQ ID NO:26. In some embodiments, an Fc domain is amino acids 1-239or 2-239 of SEQ ID NO:27. In some embodiments, an Fc domain is a humanFc domain. In some embodiments, an Fc domain is from an immunoglobin,e.g., an IgG such as IgG4. In some embodiments, an Fc domain is capableof forming a dimer with another Fc domain. In some embodiments, acomplement factor B analog of the invention is capable of forming dimers(e.g., homologous dimers), such as, but not limited to, throughinteractions of Fc domains. In some embodiments, a complement factor Banalog does not form dimers or the majority of a complement factor Banalog population/preparation is in the form of monomers. In someembodiments, a complement factor B analog comprising an Fc domain doesnot form dimers or the majority of this complement factor B analogpopulation/preparation is in the form of monomers. In some embodiments,a complement factor B analog comprises an Fc domain that has one ormutations of a cysteine(s) in the Fc domain sequence, e.g., a cysteineinvolved in dimerization of the Fc domain. In some embodiments, a Fcdomain comprises a mutation of one or more cysteines corresponding toamino acids 17 and/or 20 of SEQ ID NO:27. In some embodiments, acomplement factor B analog comprises an Fc domain that has one ormutations of a free cysteine(s) in the Fc domain sequence. In someembodiments, a cysteine(s) is substituted with an amino acid selectedfrom the group consisting of histidine, isoleucine, leucine, methionine,phenylalanine, serine, threonine, tyrosine, and valine. In someembodiments, a cysteine is deleted. In some embodiments, an amino acidsequence linker is used between amino acid sequences for a complementfactor B analog and an Fc region. In some embodiments, this linker isone amino acid, e.g., arginine.

In some embodiments, a polypeptide comprises both a truncated complementfactor B analog and an Fc region such as comprising amino acidscorresponding to amino acids 26-480 of SEQ ID NO:2 and an Fc region.

The invention further provides analogs which are fragments of acomplement factor B protein or analog that contain at least a 20, 30,50, 70, 100, 150, 200, 300, 400, 480, 500, 600 or 700 amino acid portionof the complement factor B protein or analog and/or comprises 1, 2 or 3domains of the protein and have or retain one or more biologicalactivities of the wild type complement factor B protein or analog and/oracts as an inhibitor of an aspect of the complement system (either theclassical pathway, alternative pathway or both). These fragments can befurther modified by linking or fusing with another protein or fragmentsuch as an Fc to increase the stability and/or half-life of the analog.In some embodiments, an analog is fragment of a complement factor Bprotein or analog and the fragment has at least 99.5%, 99%, 98%, 95%,90%, 85% or 62% identity with the corresponding amino acid sequence of awild-type complement factor B.

The invention provides proteins comprising a fragment of a complementfactor B protein or analog, wherein the fragment has an N-terminaland/or C-terminal truncation. In some embodiments, a complement factor Banalog comprises a C-terminal truncation wherein the analog is truncatedat or after (C-terminal to) an amino acid corresponding to amino acid407, 427, 457, 477, 480, 484, 487, 507 or 527 of SEQ ID NOs:1, 2 or 4.In some embodiments, a complement factor B analog comprises a C-terminaltruncation wherein the analog is truncated at an amino acid betweenamino acids corresponding to amino acids 407-487, 470-495 or 477-487 ofSEQ ID NOs:1, 2 or 4. In some embodiments, a complement factor B analogof the invention does not comprise amino acids corresponding to aminoacids 408-764, 428-764, 458-764, 478-764, 481-764, 485-764, 488-764,507-764, 527-764 of SEQ ID NOs:1, 2 or 4. In some embodiments,truncation or fragmentation of a factor B protein or analog can create afree cysteine. For example, a truncation can result in the deletion ofone cysteine of a cysteine pair that forms a disulfide bond in a nativecomplement factor B protein, thus creating a polypeptide that contains acysteine, but does not contain its native cysteine “partner”. In some ofthese embodiments, the remaining cysteine of the pair may be mutated,e.g., substituted with another amino acid, such as an alanine,histidine, isoleucine, leucine, methionine, phenylalanine, serine,threonine, tyrosine and valine or the cysteine may be deleted, e.g., toeliminate possible undesired disulfide bonding.

Analogs of the invention can be prepared by various techniques,including but not limited to, chemical synthesis or by expression of therecombinant analog.

Nucleotide sequences for genes and coding regions encoding human factorB, as well as the amino acid sequences are known in the art. Forexample, a gene encoding human factor B is found in NCBI DatabaseAccession No. NG_000013. A coding sequence for a human factor B is foundin NCBI Database Accession No. NM_001710 and an amino acid sequence fora human complement factor B preproprotein is found in NCBI DatabaseAccession No. NP_001701 or P00751. Sequences from other animal speciesare also known in the art. By way of comparison, in the mouse factor Bprotein sequence (e.g., see NCBI Database Accession No. P04186), thethird SCR domain is located at positions 160-217 of this 761 amino acidpreprotein, and the mature murine factor B protein spans positions23-761. The first 22 amino acids of mouse factor B protein comprises asignal sequence.

Typically, a human factor B preprotein is a 764 amino acid protein(e.g., see SEQ ID NO:1) with a signal peptide spanning amino acidpositions 1-25. The mature chain of factor B corresponds to positions26-764 (e.g., see SEQ ID NO:1). The three SCR regions of human factor Bare SCR1, also known as Sushi 1, spanning from about position 35 toabout position 100, SCR2, also known as Sushi 2, spanning from aboutposition 101 to about position 160 and SCR3, also known as Sushi 3,spanning from about position 163 to about position 220.

PCT Publication No. WO08/106644 and U.S. Patent Publication No.US20100120665 describe, inter alia, three specific dominant negativehuman factor B protein analogs designated as hfB1, hfB2 and hfB3. Thefirst of these three analogs, termed fB1, contains a mutated amino acidin the factor B (fB) protease site. This fB moiety binds C3b with normalaffinity and kinetics, but when acted upon by factor D (fD) andstabilized by properdin, does not function as a protease and does notform a C3 convertase. fB1 contains a substitution with N at an aminoacid corresponding to amino acid 740 of SEQ ID NO:1 (e.g., D740N). Thesecond of these complement factor B analogs, termed fB2, alters the sameamino acid as fB1, but in addition, alters two additional amino acids inthe C3b binding domain (substitutions at amino acids corresponding toamino acids 279 and 285 of SEQ ID NO:1) which increase the bindingaffinity of fB2 to C3b, e.g., D279G, N285D and D740N changes. The N285Dsubstitution removes a putative N-glycosylation site. The third of thesecomplement factor B protein analogs, termed hfB3, combines the mutationsthat increase C3b binding from fB2 with a mutation that knocks out thesite for cleavage by factor D, particularly with substitutions at aminoacids corresponding to residues 258, 259 and 260 of SEQ ID NO:1 as wellas substitutions at amino acids corresponding to residues 279 and 285,e.g., K258A, R259A, K260A, D279G and N285D changes. Cleavage of wildtype fB by factor D activates the fB protease. Thus, hfB3, with its fiveamino acid changes, efficiently binds C3b but has minimal proteaseactivity.

hfB1, hfB2 and hfB3 are examples of human factor B analogs which can befurther modified to complement factor B analogs of the present inventionby mutating a free cysteine corresponding to amino acid 292 of SEQ IDNO:1, e.g., by substituting the cysteine with a serine, but theinvention is not limited to these specific analogs. Some embodiments ofthe invention include any complement factor B analog that modulates acomplement pathway and does not comprise a free cysteine. In someembodiments, a complement factor B analog comprises, in addition to amutated free cysteine, one or more mutations of amino acidscorresponding to one or more of the following amino acids in SEQ IDNO:1: amino acid 258, 259, 260, 279, 285, 739, 740, 741, 742, 743, 744,745 and 746. These one or more mutations can be a substitution ordeletion of the amino acid or an addition of at least one amino acidnext to or within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of thecorresponding amino acids. In some embodiments, this addition disrupts,changes, enhances or inhibits the function of the listed amino acid,e.g., disrupts its role (i) in cleavage of another protein (e.g., 740),(ii) as a site of cleavage by another protein (e.g., amino acidscorresponding to residues 258, 259 and/or 260 of SEQ ID NO:1), or (iii)its role in binding another protein (e.g., amino acids corresponding toresidues 279 or 285 of SEQ ID NO:1).

Some embodiments of the invention comprise a substitution of an aminoacid corresponding to one or more of amino acids corresponding to 258,259 and/or 260 of SEQ ID NO:1, e.g., with an amino acid selected fromthe group consisting of alanine, glycine, valine, leucine andisoleucine. Some embodiments of the invention comprise a deletion of anamino acid corresponding to one, two or three of amino acidscorresponding to amino acids 258, 259 and/or 260 of SEQ ID NO:1. Someembodiments of the invention comprise at least one addition of 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more amino acids immediately next to an aminoacid corresponding to amino acids 258, 259 and/or 260 of SEQ ID NO:1.

Some embodiments of the invention comprise a substitution of an aminoacid corresponding to amino acid 739 of SEQ ID NO:1. Some embodiments ofthe invention comprise a substitution of an amino acid corresponding toamino acid 739 of SEQ ID NO:1, e.g., with an amino acid selected fromthe group consisting of alanine, glycine, valine, leucine andisoleucine. Some embodiments of the invention comprise a deletion of anamino acid corresponding to the 739 amino acid of SEQ ID NO:1.

Some embodiments of the invention comprise a substitution of an aminoacid corresponding to amino acid 740 of SEQ ID NO:1, e.g., with an aminoacid selected from the group consisting of glutamic acid, asparagine,alanine, serine, glycine and tyrosine. Some embodiments of the inventioncomprise a substitution of an amino acid corresponding to amino acid 740of SEQ ID NO:1 with an amino acid selected from the group consisting ofvaline, leucine, isoleucine, threonine, cysteine, methionine, glutamine,phenylalanine, tyrosine, tryptophan, glutamic acid, asparagine, alanine,serine, glycine and tyrosine. Some embodiments of the invention comprisea deletion of an amino acid corresponding to the 740 amino acid of SEQID NO:1.

Some embodiments of the invention comprise a substitution of an aminoacid corresponding to amino acid 741 of SEQ ID NO:1, e.g., with an aminoacid selected from the group consisting of tryptophan and alanine. Someembodiments of the invention comprise a substitution of an amino acidcorresponding to amino acid 741 of SEQ ID NO:1 with an amino acidselected from the group consisting of alanine, glycine, valine, leucineand isoleucine. Some embodiments of the invention comprise asubstitution of an amino acid corresponding to amino acid 741 of SEQ IDNO:1 with an amino acid selected from the group consisting oftryptophan, tyrosine and phenylalanine. Some embodiments of theinvention comprise a deletion of an amino acid corresponding to the 741amino acid of SEQ ID NO:1.

Some embodiments of the invention comprise a substitution of an aminoacid corresponding to amino acid 742 of SEQ ID NO:1, e.g., with aglutamine. Some embodiments of the invention comprise a substitution ofan amino acid corresponding to amino acid 742 of SEQ ID NO:1 with anamino acid selected from the group consisting of glutamine, glutamicacid, asparagine, and aspartic acid. Some embodiments of the inventioncomprise a deletion of an amino acid corresponding to amino acid 742 ofSEQ ID NO:1.

Some embodiments of the invention comprise a substitution of an aminoacid corresponding to amino acid 743 and/or 745 of SEQ ID NO:1, e.g.,with a phenylalanine. Some embodiments of the invention comprise asubstitution of an amino acid corresponding to amino acid 743 and/or 745of SEQ ID NO:1 with an amino acid selected from the group consisting ofphenylalanine, tyrosine and tryptophan. Some embodiments of theinvention comprise a deletion of one or more of amino acidscorresponding to amino acids 743, 744 and/or 745 of SEQ ID NO:1.

Some embodiments of the invention comprise a substitution of an aminoacid corresponding to amino acid 746 of SEQ ID NO:1, e.g., with an aminoacid selected from the group consisting of tryptophan and alanine. Someembodiments of the invention comprise a substitution of an amino acidcorresponding to amino acid 746 of SEQ ID NO:1 with an amino acidselected from the group consisting of alanine, glycine, valine, leucineand isoleucine. Some embodiments of the invention comprise asubstitution of an amino acid corresponding to amino acid 746 of SEQ IDNO:1 with an amino acid selected from the group consisting oftryptophan, tyrosine and phenylalanine. Some embodiments of theinvention comprise a deletion of an amino acid corresponding to aminoacid 746 of SEQ ID NO:1.

Some embodiments of the invention comprise the insertion or substitutionof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids immediately next toor in place of any one or more of the amino acids corresponding to aminoacids 739, 740, 741, 742, 743, 744, 745 and/or 746 of SEQ ID NO:1.

Some embodiments of the invention comprise a substitution of an aminoacid corresponding to amino acid 279 of SEQ ID NO:1, e.g., with an aminoacid selected from the group consisting of glycine, alanine andasparagine. Some embodiments of the invention comprise a substitution ofan amino acid corresponding to amino acid 279 of SEQ ID NO:1 with anamino acid selected from the group consisting of glycine, alanine,valine, leucine and isoleucine. Some embodiments of the inventioncomprise a substitution of an amino acid corresponding to amino acid 279of SEQ ID NO:1 with an amino acid selected from the group consisting ofasparagine, glutamic acid and glutamine. Some embodiments of theinvention comprise a deletion of an amino acid corresponding to aminoacid 279 of SEQ ID NO:1. Some embodiments of the invention comprise theinsertion or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more aminoacids immediately next to or in place of an amino acid corresponding toamino acid 279 of SEQ ID NO:1.

Some embodiments of the invention comprise a substitution of an aminoacid corresponding to amino acid 285 of SEQ ID NO:1, e.g., with an aminoacid selected from the group consisting of alanine and aspartic acid.Some embodiments of the invention comprise a substitution of an aminoacid corresponding to amino acid 285 of SEQ ID NO:1 with an amino acidselected from the group consisting of glycine, alanine, valine, leucineand isoleucine. Some embodiments of the invention comprise asubstitution of an amino acid corresponding to amino acid 285 of SEQ IDNO:1 with an amino acid selected from the group consisting of asparticacid, glutamic acid and glutamine. Some embodiments of the inventioncomprise a deletion of the an amino acid corresponding to amino acid 285of SEQ ID NO:1. Some embodiments of the invention comprise the insertionor substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsimmediately next to or in place of an amino acid corresponding to aminoacid 285 of SEQ ID NO:1.

Some embodiments of the invention comprise a substitution of the one ormore of the amino acids corresponding to amino acids 279, 282, 283, 284and 285 of SEQ ID NO:1. In some embodiments, these amino acids arereplaced with glycine, isoleucine, proline, histidine and aspartic acid,respectively.

Some embodiments of the invention comprise mutations of the amino acidscorresponding to 258, 259, 260, 279 and 285 of SEQ ID NO:1 as describedherein.

In some specific embodiments, the invention provides a factor B proteinanalog, that comprises the amino acid sequence of SEQ ID NO:2, 3, 21,22, or 23 (optionally without any signal sequence, e.g., amino acids1-25 contained therein). These factor B protein analogs can be used inmethods of the invention.

The invention includes complement factor B protein analogs that comprisea combination of the mutations (substitutions, deletions and additions)discussed herein. In some embodiments, these complement factor B analogsretain one or more of the attributes of the hfB1, hfB2, hfB3, hfB3-2925or hfB3-2925-740N analogs or any other complement factor B analogdiscussed herein. The invention further provides analogs that arefragments (for example comprising one or more domains of a complementfactor B protein having one or more of the amino acid mutations setforth herein) of these analogs that have one or more of the attributesof the analogs discussed above, e.g., hfB3-292SN480. In addition, theanalogs may comprise additional amino acid substitutions, deletions orinsertions (for example, conservative amino acid substitutions,truncations of the N-terminus or C-terminus, etc.) such that the analoghas at least 99.5%, 99%, 98%, 95%, 90%, 85%, 80%, 75% or 75% identitywith the corresponding wild-type complement factor B or, in the case ofan analog comprising a fragment of complement factor B, the analog hasat least 99.5%, 99%, 98%, 95%, 90%, 85%, 80%, 75% or 70% identitybetween the corresponding parts of the analog and wild-type complementfactor B.

The hfB3-292S and hfB3-292SN480 proteins contain the following keyfeatures from N-terminus to C-terminus: 1) factor B protein nativesignal sequence for efficient secretion out of mammalian expressioncells such as human 293 cells, CHO cells, BHK cells; 2) a mutatedC3b-dependent factor D protein cleavage site (changing amino acids fromK258R259K260 to A258A259A260); 3) a mutated C3b protein binding site(changing amino acid from D279 to G279; and amino acid from N285 toD285) to enable its tight binding with and trapping of C3b protein; and4) a mutated free cysteine (changing amino acid C292 to 5292) toincrease the amount of “active” or correctly folded protein.

In some embodiments of the invention, a human complement factor Bprotein analog is at least 90%, at least 95%, at least 98% or at least99% identical to (i) SEQ ID NOs:1, 2, 3, 22 or 23; (ii) amino acids26-764 of SEQ ID NOs:1, 2 or 3; (iii) amino acids 1-990 or 26-990 ofeither of SEQ ID NOs:22 or 23; amino acids 26-480 of SEQ ID NO:2; and(iv) amino acids 1-1003 or 26-1003 of SEQ ID NO:26. In some embodiments,a human complement factor B protein analog of the invention comprises(i) SEQ ID NOs:2, 3, 22, 23 or 26; (ii) amino acids 1-480 of SEQ IDNO:2; (iii) amino acids 26-480 of SEQ ID NO:2; (iv) amino acids 26-764of SEQ ID NOs:2 or 3; (v) amino acids 26-990 of SEQ ID NOs:22 or 23; or(vi) amino acids 26-1003 of SEQ ID NO:26. In some embodiments, acomplement factor B protein analog consists essentially of (i) aminoacids 26-764 of SEQ ID NO:2 or 3; (ii) amino acids 26-480 of SEQ ID NO:2or 3; (iii) amino acids 26-990 of SEQ ID NOs:22 or 23; or (iv) aminoacids 26-1003 of SEQ ID NO:26.

FIG. 1 depicts the classical and lectin complement pathways (FIG. 1a )and the alternative complement pathway (FIG. 1b ). Both pathways utilizeC3b. The C3bBb complex is a C3 convertase which converts C3 to C3b.Spontaneous dissociation (“decay”) of the C3bBb within minutes leads toits inactivation, whereas properdin stabilizes the C3bBb complex. C3bparticipates in the C3 convertase to generate additional C3b therebycreating a positive feedback loop as shown by the large arrow. Severalof the factors that attenuate the complement pathways, such as decayaccelerating factor (DAF), do so by accelerating the decay of the C3convertase, C3bBb. Without wishing to be bound by theory, some of thecomplement factor B analogs described herein bind C3b in place of anative complement factor B, thereby competing with native complementfactor B for binding to C3b. Some complement factor B analogs of theinvention bind C3b to create an inactive complex or a complex withsignificantly reduced C3 convertase activity as compared to a nativeC3bBb complex.

The invention also provides complement factor B analogs comprisingmutations of amino acids corresponding to those in complement factor Bthat interact with factors/molecules that accelerate the decay of theC3bBb complex. In some embodiments, a complement factor B analogcomprises mutations of amino acids that interact with DAF. Thesemutations include, but are not limited to, one or more mutations ofamino acids corresponding to amino acids 290, 291, 323, 363, 364, or 407of SEQ ID NO:1. In some embodiments, these mutations are a substitutionor deletion of one or more of the amino acids corresponding to aminoacids 290, 291, 323, 363, 364, or 407 of SEQ ID NO:1. In someembodiments, a complement factor B analog comprises one or more of thefollowing mutations corresponding to K323E, K290A, K291A, Y363A, S364Aor D407N of SEQ ID NO:1. In some embodiments, a complement factor Banalog comprises one of the following combinations of mutationscorresponding to K290A/K291A, Y363A/S364A or K290A/K291A/Y363A/S364A ofSEQ ID NO:1. Without wishing to be bound by theory, mutations of aminoacids in a complement factor B analog that interact withfactors/molecules that accelerate the decay of the C3bBb complex, mayinhibit the decay of complexes of C3b and a complement factor B analogof the invention, thereby allowing for a complement factor B analog tobetter inhibit complement activity.

Exemplary procedures for generating cDNAs (e.g., human wild type fB andthree complement factor B analogs as well as four analogous murinesequences) and their incorporation into vectors are detailed in Example8 of PCT Publication No. WO08/106644 and U.S. Patent Publication No.US20100120665.

Nucleic Acids

The invention includes nucleic acids comprising a nucleotide sequenceencoding a complement factor B protein analog of the invention andincludes vectors comprising these nucleic acids.

To ensure local and long term expression of a nucleic acid of interest,some embodiments of the invention contemplate transducing a cell with anucleic acid or vector encoding a complement factor B analog of theinvention. The instant invention is not to be construed as limited toany one particular nucleic delivery method, and any available nucleicacid delivery vehicle with either an in vivo or in vitro nucleic aciddelivery strategy, or the use of manipulated cells (such as thetechnology of Neurotech, Lincoln, R.I., e.g., see U.S. Pat. Nos.6,231,879; 6,262,034; 6,264,941; 6,303,136; 6,322,804; 6,436,427;6,878,544) as well as nucleic acids of the invention encoding acomplement factor B analog per se (e.g., “naked DNA”), can be used inthe practice of the instant invention. Various delivery vehicles, suchas vectors, can be used with the present invention. For example, viralvectors, amphitrophic lipids, cationic polymers, such aspolyethylenimine (PEI) and polylysine, dendrimers, such as combburstmolecules and starburst molecules, nonionic lipids, anionic lipids,vesicles, liposomes and other synthetic nucleic acid means of genedelivery (e.g., see U.S. Pat. Nos. 6,958,325 and 7,098,030; Langer,Science 249:1527-1533 (1990); Treat et al., in “Liposomes” in “TheTherapy of Infectious Disease and Cancer”; and Lopez-Berestein & Fidler(eds.), Liss, New York, pp. 317-327 and 353-365 (1989); “naked” nucleicacids and so on can be used in the practice of the instant invention.

A vector is a means by which a nucleic acid of interest (e.g., atherapeutic nucleic acid, e.g., that can encode a therapeutic protein)is introduced into a target cell of interest. A vector is typicallyconstructed or obtained from a starting material, such as a nucleic acidcapable of carrying a foreign gene or transgene and which is capable ofentering into and being expressed in a target cell. Suitable startingmaterials from which a vector can be obtained include transposons,plasmids, viruses, PCR products, cDNAs, mRNAs and so on, as known in theart. Methods for obtaining or constructing a vector of interest include,but are not limited to, standard gene manipulation techniques,sequencing reactions, restriction enzymes digests, polymerase reactions,PCR, PCR SOEing, ligations, recombinase reactions (e.g., Invitrogen'sGATEWAY® technology) other enzymes active on nucleic acids, bacteria andvirus propagation materials and methods, chemicals and reagents, sitedirected mutagenesis protocols and so on, as known in the art, see, forexample, the Maniatis et al. text, “Molecular Cloning.”

Nucleic acids of the invention will typically comprise a promoteroperatively linked to a complement factor B protein analog codingsequence. A promoter may be a tissue specific promoter, a cell specificpromoter, an inducible promoter, a repressible promoter, a constitutivepromoter, a synthetic promoter or a hybrid promoter, for example.Examples of promoters useful in the constructs of the invention include,but are not limited to, a phage lambda (PL) promoter; an SV40 earlypromoter; a herpes simplex viral (HSV) promoter; a cytomegalovirus (CMV)promoter, such as the human CMV immediate early promoter; atetracycline-controlled trans-activator-responsive promoter (tet)system; a long terminal repeat (LTR) promoter, such as a MoMLV LTR, BIVLTR or an HIV LTR; a U3 region promoter of Moloney murine sarcoma virus;a Granzyme A promoter; a regulatory sequence(s) of the metallothioneingene; a CD34 promoter; a CD8 promoter; a thymidine kinase (TK) promoter;a B19 parvovirus promoter; a PGK promoter; a glucocorticoid promoter; aheat shock protein (HSP) promoter, such as HSP65 and HSP70 promoters; animmunoglobulin promoter; an MMTV promoter; a Rous sarcoma virus (RSV)promoter; a lac promoter; a CaMV 35S promoter; and a nopaline synthetasepromoter. In some embodiments, a promoter is an MND promoter (Robbins etal., 1997, J. Virol. 71:9466-9474), or an MNC promoter, which is aderivative of the MND promoter in which the LTR enhancers are combinedwith a minimal CMV promoter (Haberman et al., J. Virol.74(18):8732-8739, 2000).

In some embodiments, a vector of the invention comprises an intron,e.g., as part of the gene coding for a complement factor B proteinanalog. Heterologous introns are known and non-limiting examples includea human β-globin gene intron. An intron can be from a complement factorB gene or a heterologous intron.

Signal sequences or leader sequences are known and can be used incomplement factor B analog expression constructs. Signal sequences aretranslated in frame as a peptide attached to the amino-terminal end of apolypeptide of choice, the secretory signal sequence will cause thesecretion of the polypeptide by interacting with the machinery of thehost cell. As part of the secretory process, this secretory signalsequence will be cleaved off. The human placental alkaline phosphatasesecretory signal sequence is an example of a useful signal sequence. Thepresent invention is not limited by specific secretory signal sequencesand others are known to those skilled in the art. The term “signalsequence” also refers to a nucleic acid sequence encoding the secretorypeptide. If a signal sequence is included, it can either be a wild typecomplement factor B sequence, a homologous sequence, or a heterologoussequence.

Viral Vectors

The present invention includes viral vectors encoding a complementfactor B analog(s) of the invention. Examples of viral vectors useful inthe present invention are described in PCT Publication No. WO08/106644and U.S. Patent Publication No. US20100120665. The present invention isnot limited to a particular viral vector. Viral vectors include, but arenot limited to, retroviral vectors, lentiviral vectors, adenoviralvectors (see, for example, U.S. Pat. No. 7,045,344), AAV vectors (e.g.,see U.S. Pat. No. 7,105,345), Herpes viral vectors (e.g., see U.S. Pat.Nos. 5,830,727 and 6,040,172), hepatitis (e.g., hepatitis D) viralvectors (e.g., see U.S. Pat. No. 5,225,347), SV40 vectors, EBV vectors(e.g., see U.S. Pat. No. 6,521,449) and Newcastle disease virus vectors(e.g., see U.S. Pat. Nos. 6,146,642, 7,442,379, 7,332,169 and6,719,979). In some embodiments, a lentiviral vector is an HIV, EIAV,SIV, FIV or BIV vector. The invention also provides a cell that producesa viral vector of the invention.

Examples of BIV systems are described, for example, in Matukonis et al.,2002 Hum. Gene Ther. 13, 1293-1303; Molina et al., 2002 Virology. 304,10-23; Molina et al., 2004 Hum. Gene Ther., 15, 65-877; U.S. Pat. Nos.6,864,085, 7,125,712, 7,153,512; PCT Publication No. WO08/106644 andU.S. Patent Publication No. US20100120665.

Vector virions of the invention may be administered in vivo or in vitroto cells (e.g., mammalian cells). Vectors (viral or nonviral) can beused to transduce or transform cells including, but not limited to,undifferentiated cells, differentiated cells, somatic cells, primitivecells and/or stem cells. In some embodiments, stem cells are intendedfor administration to a human and not for implantation in a suitablypseudopregnant woman for differentiation and development into an infant.

In some embodiments, a viral vector of the invention comprises a decayaccelerating factor (DAF). For example, an enveloped viral vectorincludes a DAF on the viral membrane. In some embodiments, a DAF is awild-type DAF. In some embodiments, a DAF is part of a fusion proteinwith an envelope protein, e.g., see Guibinga et al. Mol Ther. 200511(4):645-51. In some embodiments, a BIV producer cell expresses a DAF.

Production of Complement Factor B Analogs of the Invention

The complement factor B protein analogs of the invention can be producedfrom a cell. In some embodiments, the complement factor B protein analogis then purified from the cell and/or from cell culture medium.

The invention includes (i) cells comprising a nucleic acid comprising anucleotide sequence encoding a complement factor B analog of theinvention and/or (ii) cells expressing a complement factor B proteinanalog of the invention. In some embodiments, a mammalian cell isutilized. In some embodiments, a prokaryotic cell is utilized.

Host cells are typically transfected or transduced with an expression orcloning vector for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, amplifying the genes encoding the desired sequences orfor downstream purification and/or concentration procedures.

Suitable host cells for cloning or expressing a coding region in avector are prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes for this purpose include, but are not limited to,eubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as wellas Bacilli such as B. subtilis and B. licheniformis (e.g., B.licheniformis 41P), Pseudomonas such as P. aeruginosa, and Streptomyces.In some embodiments, an E. coli cloning host is E. coli 294 (e.g., ATCC31,446), although other strains such as E. coli B, E. coli X1776 (e.g.,ATCC 31,537), and E. coli W3110 (e.g., ATCC 27,325) may be suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts. Saccharomycescerevisiae, or common baker's yeast, is commonly used among lowereukaryotic host microorganisms. However, a number of other genera,species, and strains are commonly available and useful herein, such asSchizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis,K. fragilis (e.g., ATCC 12,424), K. bulgaricus (e.g., ATCC 16,045), K.wickeramii (e.g., ATCC 24,178), K. waltii (e.g., ATCC 56,500), K.drosophilarum (e.g., ATCC 36,906), K. thermotolerans, and K. marxiamis;yarrowia (e.g., EP402,226); Pichia pastoris (e.g., EP183,070); Candida;Trichoderma reesia (e.g., EP244,234); Neurospora crassa; Schwanniomycessuch as Schwanniomyces occidentalis; and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts suchas A. nidulans and A. niger.

Suitable host cells, e.g., for the expression of glycosylated proteins,can be derived from multicellular organisms. Examples of invertebratecells include plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified and can be used for expressing proteins. Avariety of viral strains for transfection can be used for proteinexpression and are publicly available, e.g., the L-1 variant ofAutographa californica NPV and the Bm-5 strain of Bombyx mori NPV, andsuch viruses may be used according to the present invention, forexample, for transfection of Spodoptera frugiperda cells. Plant cellcultures of cotton, corn, potato, soybean, petunia, tomato, and tobaccocan also be utilized as hosts.

Some embodiments of the invention utilize vertebrate or mammalian cells,and propagation of vertebrate cells in culture (tissue culture) can be aroutine procedure. Examples of useful mammalian host cell lines are amonkey kidney CVI cell line transformed by SV40 (e.g., COS-7, ATCC CRL1651); human embryonic kidney line (e.g., 293 or 293T cells includingeither cell line subcloned for growth in suspension culture, Graham etal., J. Gen Virol. 36:59 (1977) such as 293 Freestyle (Invitrogen,Carlsbad, Calif.)) or 293FT; baby hamster kidney cells (e.g., BHK, ATCCCCL 10); Chinese hamster ovary cells; Chinese hamster ovary cells/-DHFR(e.g., CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980));mouse sertoli cells (e.g., TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (e.g., CVI ATCC CCL 70); African greenmonkey kidney cells (e.g., VERO-76, ATCC CRL-1587); human cervicalcarcinoma cells (e.g., HELA, ATCC CCL 2); canine kidney cells (e.g.,MOCK, ATCC CCL 34); CF2TH cells; buffalo rat liver cells (e.g., BRL 3A,ATCC CRL 1442); human lung cells (e.g., W138, ATCC CCL 75); human livercells (e.g., Hep G2, HB 8065); mouse mammary tumor cells (e.g., MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1983)); MRC 5 cells; and FS4 cells.

In some instances, a host cell may be modified to decrease or eliminateexpression of an endogenous protein. For example, if a complement factorB protein analog is to be produced in a particular host cell (e.g., aCHO cell), then the host cell could be modified so as expression of thehost cell's native factor B protein (e.g., hamster factor B) is reducedor eliminated. This may be advantageous for the downstream purificationof the complement factor B protein analog. Therefore, the inventionprovides a method of producing a complement protein analog of theinvention comprising reducing or eliminating the expression of thecorresponding native complement protein in the host cell. Methods forreducing, eliminating or knocking out expression of a host cell proteinare known in the art. For example, a protein's expression level may bereduced or eliminated by engineering the host cell to express inhibitoryRNA (e.g., RNAi) specific for the RNA coding for the protein. Forexample, Clontech (Mountain View, Calif.) sells various vectors andkits, such as those referred to as part of the KNOCKOUT™ RNAi Systems,for knocking down expression of proteins in a host cell. Other methodsinclude gene targeting by homologous recombination which allows theintroduction of specific mutations into any cloned gene, e.g., seeCurrent Protocols in Molecular Biology, John Wiley & Sons, Inc.,1994-1998, Sections 9.16 and 9.17. This can be used to knockout the geneexpressing the host cell protein.

Another method which may be utilized to reduce expression of anendogenous protein, involves using a targeted transcription factor thatrepresses expression of the endogenous protein. For example, a repressordomain from a transcription factor may be attached or fused to a DNAbinding domain such as a zinc finger polypeptide. One skilled in the artcan design zinc finger polypeptides that bind specific DNA sequences,e.g., see U.S. Pat. Nos. 6,140,081; and 7,067,617; and U.S. PublishedPatent Applications 20060078880; 20040224385; and 20070213269. Oneskilled in the art can associate designed zinc finger polypeptides witha transcriptional repressor domain (e.g., a KRAB (Krüppel-associatedbox) domain). Examples of such molecules and techniques are described inBeerli et al. (Proc Natl Acad Sci USA. 2000 97(4):1495-4500) and U.S.Published Patent Application 20070020627. In some embodiments of theinvention, a host cell would be transduced with a vector expressing thetranscriptional repressor. This approach has an advantage over knockingout the gene of interest using homologous recombination because, in mostcases, a host cell will be diploid and it would be desirable to knockout both gene copies. Whereas, expression of a transcriptional repressorshould repress expression of both gene copies.

The expression of particular endogenous protein may also be reducedusing compounds that will directly or indirectly reduce the expressionof the particular endogenous protein. Using fB as an example, variouscompounds can be used to reduce the expression of endogenous fBexpression. For example, fB protein expression has been shown to beinhibited by histamine (Falus & Meretey, Immunology 1987 60:547-551 andFalus & Meretey, Mol Immunol 1988 25(11):1093-97), sodium butyrate(Andoh et al. Clin Exp Immuno 1999 118:23-29), a glucocorticoid such asdexamethasone (Dauchel et al. Eur J Immunol 1990 20(8):1669-75),platelet derived growth factor (Circolo et al. 1990 The Journal of BiolChem 265(9):5066-5071), epidermal growth factor (Circolo et al. 1990),and fibroblast growth factor (Circolo et al, 1990). A host cell of theinvention may be cultured in the presence of any one or combination ofthese molecules to reduce the endogenous expression of complement factorB protein. Therefore, in some embodiments of the invention, a host cellexpressing a complement factor B protein analog is cultured in thepresence of any one or more compounds selected from the group consistingof a histamine, a sodium butyrate, a glucocorticoid (e.g.,dexamethasone), a platelet derived growth factor, an epidermal growthfactor, or a fibroblast growth factor.

Various compounds and proteins have been shown to upregulate or maintainexpression of complement factor B protein. For example, complementfactor B protein expression has been shown to be upregulated ormaintained by tumor necrosis factor (TNF) (Andoh et al. Clin Exp Immuno1999 118:23-29), estrogen (Sheng-Hsiang et al. Biology of Reproduction2002 66:322-332), Interleukin-1 (Dauchel et al. Eur J Immunol 199020(8):1669-75), dexamethasone (Lappin & Whaley, Biochem J 1991280:117-123), prednisolone (Lappin & Whaley 1991), cortical (Lappin &Whaley 1991), and Interferon-gamma (Huang et al. 2001 Eur J Immunol31:3676-3686). A host cell of the invention may be cultured in theabsence of any one or combination of these molecules to reduce theendogenous expression of complement factor B protein. Additionally, ahost cell may be cultured in the presence of an inhibitor of any one ormore of these compounds. Therefore, in some embodiments of theinvention, a host cell expressing an complement factor B protein analogis cultured in the presence of any one or more compounds that inhibit acompound selected from the group consisting of a TNF, estrogen,interleukin-1, dexamethasone, prednisolone, cortical, andinterferon-gamma. In some embodiments, expression by the host cell ofone or more of these compounds is reduced, e.g., using methods asdescribed herein. Examples of inhibitors of estrogen include, but arenot limited to, tamoxifen. Inhibitors also include antibodies that bindand reduce the activity of the compound. For example, various antibodiesthat bind and inactivate TNF are know in the art.

A complement factor B protein analog containing composition preparedfrom cells can be purified using, for example, hydroxylapatitechromatography, gel electrophoresis, dialysis, size exclusionchromatography, affinity chromatography, immunoaffinity chromatography,tangential flow purification, diafiltration, ion exchangechromatography, hydrophobic interaction chromatography (HIC), reversephase chromatography, heparin sepharose affinity chromatography andother known forms of separation and concentration. Following anypreliminary purification step(s), a mixture comprising a complementfactor B protein analog and contaminants, if any, may be subjected tolow pH hydrophobic interaction chromatography, e.g., using an elutionbuffer at a pH between about 2.5-4.5, in some cases performed at lowsalt concentrations (e.g., from about 0-0.25M salt) or other proceduresfor further purification.

In some embodiments, a complement factor B protein analog of theinvention is at least 90%, at least 93%, at least 95%, at least 98%, atleast 99.5% or at least 99.9% pure in relation to total protein.

The invention also provides methods of producing a complement factor Bprotein analog comprising expressing in a cell a complement factor Bprotein analog of the invention and purifying the complement factor Bprotein analog.

Complement Mediated Conditions/Diseases

There are three pathways of complement activation, the classicalpathway, the alternative pathway, and the lectin pathway (FIG. 1).Described herein are examples of complement factor B protein analogs ofthe invention. In some embodiments, these complement factor B proteinanalogs can attenuate the alternative pathway of complement activation.However, based on the way all three complement pathways intersect, theseanalogs can diminish inflammation caused by any of the three complementpathways and thereby provide therapy for any illness whose etiologyinvolves, at least in part, complement activation. These include, butare not limited to, early AMD, wet AMD, and geographic atrophy. FIGS. 1Aand 1B outline complement pathways. Note that they intersect at C3b.

The invention provides methods of treating a complement-mediated diseasecomprising administering to a patient a pharmaceutical preparation ofthe invention, a complement factor B analog of the invention or anucleic acid or vector encoding a complement factor B analog of theinvention. The invention also includes methods of inhibiting complementactivity, wherein the method comprises administering to a human subjecta mutated human complement factor B analog in an amount sufficient toinhibit a complement pathway by competing with binding of nativecomplement factor B in the subject, wherein the mutated human complementfactor B analog is an active complement factor B analog of SEQ ID NO:4having cysteine amino acids that form disulfide bonds and a freecysteine amino acid that has been substituted by an amino acid selectedfrom the group consisting of histidine, isoleucine, leucine, methionine,phenylalanine, serine, threonine, tyrosine, and valine. In someembodiments, the mutated complement factor B analog comprises SEQ IDNO:2, 3, 22 or 23.

The invention also provides methods of inhibiting complement activity,wherein the methods comprise introducing to a site of the complementactivity a complement factor B analog of the invention, a nucleic acidof the invention, a viral vector of the invention, a pharmaceuticalcomposition/preparation of the invention, or any combination thereof inan amount sufficient to inhibit the complement activity. In someembodiments, a method of the invention utilizes a complement factor B isan analog of SEQ ID NO:4 having cysteine amino acids that form disulfidebonds and a free cysteine amino acid that has been substituted byanother amino acid.

The invention provides methods of inhibiting complement activity,wherein the methods comprise introducing to a site of the complementactivity a mutated human complement factor B analog in an amountsufficient to inhibit the complement activity by the mutated humancomplement factor B analog competing with binding of native complementfactor B, wherein the mutated human complement factor B analog is anactive complement factor B analog of SEQ ID NO:4 having cysteine aminoacids that form disulfide bonds and a free cysteine amino acid that hasbeen substituted by an amino acid. In some embodiments, this methodutilizes a complement factor B analog comprising amino acids 26-764 ofSEQ ID NO:2, amino acids 26-764 of SEQ ID NO:3, amino acids 26-990 ofSEQ ID NO:22 or amino acids 26-990 of SEQ ID NO:23.

Complement pathways are a part of the immune system known as the innateimmune system that provides immediate protection from infection prior toactivation of the humoral and cell mediated branches of the immunesystem. They are activated and inactivated through cascading reactionsthat exhibit high order kinetics but are remarkably well-regulated. Thealternative complement pathway, in particular, has evolved to cycle upwith great rapidity through a positive feedback loop.

Complement factor B protein analogs of the invention and/or the vectorsthat express them may advantageously be used for local and/or systemicadministration to a mammal and/or to treat chronic diseases. In someembodiments of the invention, a complement factor B analog inhibits acomplement pathway by competing with binding of the native complementfactor B, e.g., to C3b protein and/or factor D protein. This can allowattenuation of complement activity as opposed to complete blockade ofthe pathway. Therefore, it may be possible to downregulate complementactivity to a level that is therapeutic (e.g., alleviates some symptomsor their severity) without completely blocking complement activity.Thus, avoiding or decreasing the risks associated with blockage ofcomplement activity, such as increased risk of infection. Therefore, thepresent invention provides methods for treating a complement mediateddisease (e.g., a chronic disease) by local or systemic administration(e.g., i.v., intraperitoneal or oral) of a complement factor B proteinanalog of the invention.

In some embodiments, the present invention provides compositions andmethods for modulating, regulating, inhibiting and/or enhancing acomplement activity. Complement-related pathways include, but are notlimited to, the classical, lectin and alternative complement pathways.In some cases, a complement-related pathway may play a role in aparticular condition, disease or diseases. Therefore, some embodimentsof the invention provide methods of regulating, modifying, curing,inhibiting, preventing, ameliorating, slowing progression of and/ortreating a disease state mediated by one or more complement-relatedpathways by administering a complement factor B analog of the inventionor a nucleic acid or vector encoding a complement factor B analog of theinvention. Such disease states or conditions include, but are notlimited to, drusen formation, macular degeneration, AMD, dry eye,corneal ulcers, atherosclerosis, diabetic retinopathy, vitreoretinopathy(Grisanti et al. Invest. Ophthalmol. Vis. Sci. 32:2711-2717), cornealinflammation, airway hyperresponsiveness, immune-related diseases,autoimmune-related diseases, lupus nephritis, systemic lupuserythematosus (SLE), arthritis (e.g., rheumatoid arthritis),rheumatologic diseases, anti-phospholipid antibody syndrome, intestinaland renal I/R injury, asthma, atypical hemolytic-uremic syndrome, TypeII membranoproliferative glomerulonephritis, non-proliferativeglomerulonephritis, fetal loss (e.g., spontaneous fetal loss), glaucoma,uveitis, ocular hypertension, brain injury (e.g., traumatic braininjury), stroke (e.g., see Arumugam et al. PNAS 93(12):5872-6 (1996)),post-traumatic organ damage, thermal trauma (e.g., burn injury) postinfarction organ damage (e.g., cardiac, neurological), vasculitis,Kawasaki disease, hereditary angioedema (HAE), paroxysmal nocturnalhemoglobinuria (PNH, sometimes referred to as Marchiafava-Michelisyndrome), colitis, inflammatory bowel disease, tumor metastasis,ischemic-reperfusion injury, cerebrovascular accident, Alzheimer'sdisease, transplant rejection (e.g., xeno and allo), infections, sepsis,septic shock, Sjögren's syndrome, myasthenia gravis, antibody-mediatedskin diseases, all antibody-mediated organ-specific diseases (includingType I and Type II diabetes mellitus, thyroiditis, idiopathicthrombocytopenic purpura and hemolytic anemia, and neuropathies),insulin resistance syndrome (e.g., see Weyer et al. (2000) DiabetesCare, 23(6):779-785), gestational diabetes, multiple sclerosis,psoriasis, cardiopulmonary bypass injury, polyarteritis nodosa, HenochSchonlein purpura, serum sickness, Goodpasture's disease, systemicnecrotizing vasculitis, post streptococcal glomerulonephritis,idiopathic pulmonary fibrosis (usual interstitial pneumonitis),membranous glomerulonephritis, myocarditis (e.g., autoimmunemyocarditis) (Kaya et al. Nat Immunol. 2001; 2(8):739-45), myocardialinfarction, muscular dystrophy (e.g., associated withdystrophin-deficiency), acute shock lung syndrome, adult respiratorydistress syndrome, reperfusion, and/or a complement mediated disease.

In some embodiments, a complement-mediated disease is a disease of theeye. In some embodiments, a complement factor B analog or pharmaceuticalcomposition is administered to the eye, for example, by intravitrealinjection, subretinal injection, injection to the intraanterior chamberof the eye, injection or application locally to the cornea,subconjunctival injection, subtenon injection, or by eye drops. In someembodiments, a pharmaceutical composition is administered to the eye,wherein the pharmaceutical composition comprises at least one complementfactor B analog, e.g., selected from the group hfB3-292S (SEQ ID NO:2),hfB3-2925-740N (SEQ ID NO:3), hfB3-2925-Fc (SEQ ID NO:22) andhfB3-2925-740N-Fc (SEQ ID NO:23).

Age-related macular degeneration (AMD) is the most common cause ofdecreased vision in individuals over 65 years of age in the developedworld. Dry AMD is characterized by a progressive degeneration of themacula causing central field visual loss. A more acute debilitating AMDincludes florid neovascularization and extravasation in the retina,known as wet AMD. There is currently no effective therapy for AMD.

A characteristic of AMD is the accumulation of drusen, located betweenthe basal lamina of the retinal pigment epithelium (RPE) and the innerlayer of Bruch's membrane (Pauleikhoff et al., 1990 Am. J. Ophthalmol.109, 38-43; Bressler et al., 1990 Arch. Ophthalmol. 108, 1442-1447).Drusen, as well as other age-related changes that occur proximal toBruch's membrane, are believed to contribute to the dysfunction anddegeneration of the RPE and retina by inducing ischemia as well asrestricting the exchange of nutrient and waste products between theretina and choroid (reviewed by Bird, 1992 Pathophysiology of AMD. InAge-Related Macular Degeneration: Principles and Practice (Hampton, G.,and Nelsen, P. T., eds.) Chap. 3, Raven Press, New York). Severalstudies have indicated immune-mediated processes in the development ofAMD. Importantly, autoantibodies were detected in the sera of AMDpatients (Penfold et al., 1990 Graefes Arch. Clin. Exp. Ophthalmol. 228,270-274), as predicted by the hypothesis that immune andinflammatory-mediated processes are involved in the development and/orremoval of drusen.

The formation of drusen in the eye can be associated with variousdiseases such as macular degeneration. In some cases, drusen formationand/or its association with a disease has been implicated to be relatedto complement activity. Some embodiments of the invention providecompositions and methods for modulating, regulating, inhibiting,reducing, retarding and/or reversing the formation or growth of drusenin an animal, such as a human. For example, compositions or molecules ofthe invention may be delivered to drusen (e.g. by direct injection intodrusen (intradrusen injection), adjacent to drusen or intravitrealinjection). Some embodiments of the invention can be utilized to slowthe progression of macular degeneration, possibly by inhibiting drusenformation. Vitronectin, an abundant component of drusen, is also acomponent of extracellular deposits associated with atherosclerosis(Niculescu et al., 1989 Atherosclerosis, 78, 197-203), amyloidosis(Dahlback et al., 1993 J. Invest. Dermatol. 100, 166-170), elastosis(Dahlback et al., 1988 Acta Derm. Venereol. 68, 107-115), and MPGN typeII (Jansen et al., 1993 Am. J. Pathol. 143, 1356-1365). Vitronectin is amultifunctional protein that functions in cell adhesion, maintenance ofhemostasis, and inhibition of complement-induced cell lysis (Preissner,1991 Ann. Rev. Cell Biol. 7, 275-310). Furthermore, atheroscleroticplaques share a number of other constituents with drusen, such ascomplement components and apoliproprotein E. An association betweenadvanced AMD and atherosclerosis of carotid arteries was reported in anepidemiological study (Vingerling et al., 1995 Am. J. Epidemiol. 142,404-409) and another study identified a significant correlation betweenelastotic degeneration of nonsolar-exposed dermis and choroidalneovascularization in AMD patients (Blumenkranz et al., 1986Ophthalmology, 93, 552-558). Finally, amyloid β peptide, a majorconstituent of neuritic plaques in Alzheimer's disease, is also found indrusen (Johnson et al., 2002 Proc. Natl. Acad. Sci. USA, 99,11830-11835). Amyloid β peptide has been implicated as a primaryactivator of complement (Bradt et al., 1998 J. Exp. Med. 188, 431-438).

Comprehensive analysis of the molecular composition of human drusen, aswell as of the RPE cells that flank or overlie drusen, demonstratedimmunoreactivity to immunoglobulins and components of the complementsystem that are associated with immune complex deposition (Johnson etal., 2000 Exp. Eye Res. 70, 441-449). Drusen also containsmultifunctional proteins such as vitronectin (Hageman et al., 1999 FASEBJ. 13, 477-484) and apolipoprotein E (Anderson et al., 2001 Am. J.Ophthalmol. 131, 767-781) that play a role in immune system modulation.In addition, molecules involved in the acute phase response toinflammation, such as amyloid P component and α₁-antitrypsin, have alsobeen identified in drusen (Mullins et al., 2000 The FASEB Journal, 14,835-846), as well as proteins involved in coagulation and fibrinolysis(factor X, thrombin, and fibrinogen) (Mullins et al., 2000 The FASEBJournal, 14, 835-846). Drusen formation and associated RPE pathologywere suggested to contribute to a chronic inflammatory response thatactivates the complement cascade (Hageman et al., 2001 Prog. Retin, EyeRes. 20, 705-732; Johnson et al., 2001 Exp. Eye Res. 73, 887-896).

One other form of an optic disorder arising from AMD and resulting inperturbations of the retina is geographic atrophy, which leads to deathof patches of rod and cone cells, as well as of the RPE cells.

Atherosclerosis has been shown to typically involve complement relatedpathways, e.g., see Niculescu et al. Immunologic Research, 30(0:73-80(8)(2004) and Niculescu and Korea, Immunologic Research 30(1):73-80 (2004).Complement activation and C5b-9 deposition typically occurs both inhuman and experimental atherosclerosis. C5b-9 may be responsible forcell lysis, and sublytic assembly of C5b-9 induces smooth muscle cell(SMC) and endothelial cell (EC) activation and proliferation. ComplementC6 deficiency has a protective effect on diet-induced atherosclerosis,suggesting that C5b-9 assembly is required for, or at least plays asignificant role, in the progression of atherosclerotic lesions, e.g.,see Niculescu and Horea, Immunologic Research 30(1):73-80 (2004). Someembodiments of the invention may be used to inhibit the formation ofC5b-9 and/or inhibit atherosclerosis. In some embodiments, a complementfactor B protein analog of the invention is administered to a site orpotential site of atherosclerosis. This complement factor B proteinanalog inhibits a pathway (e.g., the classical and/or alternativecomplement pathway) which in turn inhibits the formation or activationof C5b-9 or another complement pathway related compound involved inatherosclerosis. There may be other complement related proteins involvedin atherosclerosis whose formation and/or activation may be inhibited orblocked in a similar manner.

Airway hyperresponsiveness (AHR) is characteristic of various diseasesincluding, but not limited to, asthma (e.g., allergic asthma). AHR hasbeen shown to typically involve complement related pathways, e.g., seeTaube et al., 2006 PNAS 103(21):8084-8089; Park et al., American Journalof Respiratory and Critical Care Medicine 169:726-732, (2004); Thurmanand Holers, J Immunology 176:1305-1310 (2006) and U.S. PatentPublication No. 20050260198. Park et al. showed that Crry-Igadministered by intraperitoneal injection had an effect on AHR. Someembodiments of the invention provide compositions and methods formodulating, regulating, inhibiting and/or reducing AHR in an animal,such as a human. Specific AHR related diseases that may be treated,alleviated, inhibited and/or ameliorated include, but are not limitedto, asthma, chronic obstructive pulmonary disease (COPD), allergicbronchopulmonary aspergillosis, hypersensitivity pneumonia, eosinophilicpneumonia, emphysema, bronchitis, allergic bronchitis bronchiectasis,cystic fibrosis, tuberculosis, hypersensitivity pneumonitis,occupational asthma, sarcoid, reactive airway disease syndrome,interstitial lung disease, hyper-eosinophilic syndrome, rhinitis,sinusitis, exercise-induced asthma, pollution-induced asthma, coughvariant asthma, parasitic lung disease, respiratory syncytial virus(RSV) infection, parainfluenza virus (PIV) infection, rhinovirus (RV)infection, Hantaan virus (e.g., four-corners strain) and adenovirusinfection

Immune-related diseases such as autoimmune-related diseases, HLA-B27associated inflammatory diseases, lupus nephritis and systemic lupuserythematosus (SLE) have been shown to typically involve complementrelated pathways, e.g., see Thurman and Holers, J Immunology176:1305-1310 (2006). Lupus nephritis is one complication of SLE. It isrelated to the autoimmune process of lupus, where the immune systemproduces antibodies (antinuclear antibody and others) against bodycomponents. Complexes of these antibodies and complement componentstypically accumulate in the kidneys and result in an inflammatoryresponse. Some embodiments of the invention provide methods andcompositions for regulating, modifying, curing, inhibiting, preventing,ameliorating and/or treating an immune-related disease, e.g., involvingor related to a complement pathway, such as SLE.

Arthritis has been shown to typically involve complement relatedpathways, e.g., see Thurman and Holers, J Immunology 176:1305-1310(2006) and Banda et al. J Immunol. 177(3):1904-12 (2006). Thealternative complement pathway plays a significant role in the inductionof arthritis and the alternative complement pathway may even berequired. Some embodiments of the invention provide methods andcompositions for regulating, modifying, curing, inhibiting, preventing,ameliorating and/or treating arthritis, e.g., rheumatoid arthritis orinflammatory arthritis.

Paroxysmal nocturnal hemoglobinuria (PNH) a potentially life-threateningdisease of the blood characterized by complement-induced intravascularhemolytic anemia and thrombosis due to intravascular destruction of redblood cells (RBCs) by complement resulting in uncontrolled amplificationof the complement system that leads to destruction of the RBC membrane.Persons with this disease typically have blood cells that are missing agene called PIG-A. This gene allows a substance calledglycosyl-phosphatidylinositol (GPI) to help certain proteins stick tocells. Without PIG-A, complement regulating proteins cannot connect tothe cell surface and protect the cell from complement. Some embodimentsof the invention provide methods and compositions for regulating,modifying, curing, inhibiting, preventing, ameliorating and/or treatingParoxysmal nocturnal hemoglobinuria.

Hereditary angioedema (HAE) is a potentially life-threatening geneticcondition typically caused by a deficiency of the C1 inhibitor, aprotein of the complement system. Symptoms include episodes of edema(swelling) in various body parts including the hands, feet, face andairway. Hereditary angioedema (HAE) exists in three forms, all of whichare caused by a genetic mutation that is inherited in an autosomaldominant form. Types I and II are caused by mutations in the SERPING1gene, which result in either diminished levels or dysfunctional forms ofthe C1-inhibitor protein (type I HAE). Type III HAE has been linked withmutations in the F12 gene, which encodes the coagulation protein FactorXII. All forms of HAE lead to abnormal activation of the complementsystem. Some current treatments include Ecallantide a peptide inhibitorof kallikrein (e.g., see U.S. Patent Publication No. US20070213275),Icatibant (Firazyr, Jerini) which is a selective bradykinin receptorantagonist, and Cinryze (Viropharma, Inc.) a C1 esterase inhibitor. Someembodiments of the invention provide methods and compositions forregulating, modifying, curing, inhibiting, preventing, amelioratingand/or treating Paroxysmal nocturnal hemoglobinuria.

Glaucoma is a group of diseases of the optic nerve involving loss ofretinal ganglion cells in a characteristic pattern of optic neuropathy.Approximately 25% of glaucoma patients with retinal ganglion cell losshave normal ocular pressure. Ocular hypertension (OHT) is a significantrisk factor for developing glaucoma and lowering it via pharmaceuticalsor surgery is currently the mainstay of glaucoma treatment. Ocularhypertension and glaucoma have been shown to typically involvecomplement related pathways, e.g., see Khalyfa et al., Molecular Vision,13:293-308 (2007); Stasi et al. IOVS 47(3):1024-1029 (2007); and Kuehnet al., Experimental Eye Research 83:620-628 (2006). Expression and/orthe presence of C1q and C3 have been shown to be higher in retinasubjected to OHT. Some embodiments of the invention provide methods andcompositions for regulating, modifying, curing, inhibiting, preventing,ameliorating and/or treating glaucoma.

Uveitis has been shown to typically be associated with the complementpathway, e.g., see Mondino and Rao, Investigative Ophthalmology & VisualScience 24:380-384 (1983) and Jha et al. Molecular Immunology44:3901-3908 (2007). Mondino and Rao found that mean values of alltested complement components in aqueous humor to serum measurements wereincreased in patients with a history of previous eye surgeries and werehighest in patients with anterior uveitis. Some embodiments of theinvention provide methods and compositions for regulating, modifying,curing, inhibiting, preventing, ameliorating and/or treating uveitis.

Diabetic retinopathy is one of the leading causes of vision loss inmiddle-aged individuals. Activation of the complement system is believedto play an important role in the pathogenesis of diabetic retinopathy(e.g., see Jha et al. Molecular Immunology 44:3901-3908 (2007)). Someembodiments of the invention provide methods and compositions forregulating, modifying, curing, inhibiting, preventing, amelioratingand/or treating diabetic retinopathy.

Proliferative vitreoretinopathy (PV) is one of the most commoncomplications of retinal detachment. PV has been linked to complementactivity, e.g., see Grisante et al. Invest Ophthalmol Vis Sci. 1991;32(10):2711-7 and Grisante et al. Ophthalmologe. 1992; 89(1):50-4. Someembodiments of the invention provide methods and compositions forregulating, modifying, curing, inhibiting, preventing, amelioratingand/or treating PV.

Anti-phospholipid antibody syndrome, intestinal and renal ischemicreperfusion I/R injury, atypical hemolytic-uremic syndrome, Type IImembranoproliferative glomerulonephritis, and fetal loss (e.g.,spontaneous fetal loss), have been shown to typically involve complementrelated pathways, e.g., see Thurman and Holers, J Immunology176:1305-1310 (2006).

Brain injury (e.g., traumatic brain injury) has been shown to typicallyinvolve complement related pathways, e.g., see Leinhase et al., JNeuroinflammation 4:13 (2007) and BMC Neurosci. 7:55 (2006). Leinhase2006, showed that after experimental traumatic brain injury in wild-type(fB+/+) mice, there was a time-dependent systemic complement activation.In contrast, the extent of systemic complement activation wassignificantly attenuated in fB−/− mice. Some embodiments of theinvention provide methods and compositions for regulating, modifying,curing, inhibiting, preventing, ameliorating and/or treating neuronalcell death, traumatic neural injury (e.g. brain), complement-mediatedneuroinflammation and/or neuropathology.

Ischemia-reperfusion injury can cause increases in the production of oroxidation of various potentially harmful compounds produced by cells andtissues, which can lead to oxidative damage to or death of cells andtissues. For example, renal ischemia-reperfusion injury can result inhistological damage to the kidneys, including kidney tubular damage andchanges characteristic of acute tubular necrosis. The resultant renaldysfunction permits the accumulation of nitrogenous wastes ordinarilyexcreted by the kidney, such as serum urea nitrogen (SUN).Ischemia-reperfusion may also cause injury to remote organs, such as thelung. Some embodiments of the invention utilize modulators, such asinhibitors, of a complement pathway (e.g., inhibitors of factor Bactivity), e.g., when administered to an animal that has, or is at riskof experiencing or developing, ischemia-reperfusion. In someembodiments, these modulators, prevent, reduce or inhibit at least onesymptom of injury due to ischemia-reperfusion. Other types ofischemia-reperfusion injury, that can be prevented or reduced usingmethods and compositions of the invention, include, but are not limitedto, cardiac ischemia-reperfusion injury such as myocardial infarction orcoronary bypass surgery, central nervous system ischemia-reperfusioninjury, ischemia-reperfusion injury of the limbs or digits,ischemia-reperfusion of internal organs such as the lung, liver orintestine, or ischemia-reperfusion injury of any transplanted organ ortissue. See, e.g., PCT Publication No. WO03/061765 which discussesmyocardial infarction and complement pathways.

Inflammation is a major etiologic determinant of myocardial infarction(Ridker, 2007 Nutr. Rev. 65(12 Pt 2):5253-9). It has also been shownthat delivery (e.g., intracoronary) of bone marrow (stem) cells leads toan improvement in systolic function after acute myocardial infarction(Wollert, 2008, Curr. Opin. Pharmacol. January 31 [Epub]). Also, bonemarrow stem cells can regenerate infarcted myocardium (Orlic et al. 2003Pediatr. Transplant. 7 Suppl 3:86-88). Mesenchymal stem cells have beenshown to provide a cardiac protective effect in ischemic heart disease(Guo et al. 2007 Inflammation 30(3-4):97-104). In the present invention,delivery of the stem cells can be by any means, such as intracoronaryinjection, injection directly into myocardium (e.g., into diseasedand/or healthy myocardium (e.g., adjacent to the injured area)). In someembodiments, a mammal is treated with cytokines to mobilize their bonemarrow stem cells in the circulation allowing the stem cells to trafficto the myocardial infarct.

Various stem cells have been used in vivo for various applications. Oneproblem with the use of stem cells in vivo is the lower than desiredsurvival and/or seeding of the stem cells, e.g., in the area ofinterest. One significant reason for low seeding and survival of stemscells can be inflammation at the site. Therefore, the present inventionprovides a method of treatment and/or a method of improving stem cellsurvival and/or seeding. In some embodiments, these methods compriseadministering a composition of the invention before, during and/or afteradministration or mobilization of stem cells. In some embodiments,complement inhibitors of the invention act as anti-inflammatory agentsthat will create a favorable environment for stem cells to home in andsurvive in the area of desired seeding (e.g., damaged heart or bonemarrow) and therefore repair or replace the damaged tissue. Stem cellsmay be administered in a solution that also contains a complement factorB protein analog of the invention. Stem cells may be, but are notlimited to, hematopoietic stem cells, embryonic stem cells, mesenchymalstem cells, neural stem cells, mammary stem cells, olfactory stem cells,pancreatic islet stem cells, totipotent stem cells, multipotent stemcells or pluripotent stem cells. The stem cells may be autologous,allogeneic, or syngeneic.

Complement activity appears to be involved in muscular dystrophy (e.g.,associated with dystrophin-deficiency). For example, see PCT PublicationNo. WO2007130031, Spuler & Engel 1998 Neurology 50:41-46, and Selcen etal. 2001 Neurology 56:1472-1481. Therefore, some embodiments of theinvention provide methods and compositions for regulating, modifying,curing, inhibiting, preventing, ameliorating and/or treating musculardystrophy.

Complement activity may contribute to corneal inflammation. Therefore,some embodiments of the invention provide methods and compositions forregulating, modifying, curing, inhibiting, preventing, amelioratingand/or treating corneal inflammation, e.g., after surgery. In someembodiments, a complement factor B analog of the invention isadministered via eye drops or as otherwise described herein.

In some embodiments, complement factor B analogs of the invention areused for regulating, modifying, curing, inhibiting, preventing,ameliorating and/or treating corneal neovascularization.

Some embodiments of the invention provide methods for enhancing theefficacy of post-coronary or peripheral artery bypass grafting orangioplasty. In some embodiments, a vector of the invention encoding acomplement factor B protein analog of the invention (e.g., hfB3-2925 orhfB3-2925-740N) is used to transduce cells of a blood vessel (e.g.,endothelial cells). In some embodiments, cells of a blood vessel aretransduced prior to implantation in an animal. In some embodiments,cells of a blood vessel are transduced in vivo.

Alleviating pain and suffering and inflammation in postoperativepatients is an area of special focus in clinical medicine, especiallywith the growing number of out-patient operations performed each year.Complement factor B analogs of the present invention can be utilized toinhibit inflammation, e.g., by inhibiting a complement activity.Therefore, complement factor B analogs can be used to reduceinflammation, e.g., in postoperative patients. In some embodiments, acomplement factor B analog is administered locally (e.g., perioperativedelivery) to a site of surgery to inhibit inflammation, which in somecases will reduce pain and suffering. In some embodiments, a complementfactor B analog is administered in a solution, e.g., in a physiologicelectrolyte carrier fluid. In some embodiments, a complement factor Banalog is delivered via perioperative delivery directly to a surgicalsite of an irrigation solution containing the composition. In someembodiments, due to the local perioperative delivery method of thepresent invention, a desired therapeutic effect may be achieved withlower doses of agents than are necessary when employing other methods ofdelivery, such as intravenous, intramuscular, subcutaneous and oral. Insome embodiments, when used perioperatively, the solution will result ina clinically significant decrease in operative site pain and/orinflammation, thereby allowing a decrease in the patient's postoperativeanalgesic (e.g., opiate) requirement and, where appropriate, allowingearlier patient mobilization of the operative site. In some embodiments,no extra effort on the part of the surgeon and operating room personnelis required to use the present solution relative to conventionalirrigation fluids. In some embodiments, a composition of the inventionis used (e.g., in irrigation fluid) for arthroscopy, cardiovascular andgeneral vascular therapeutic and diagnostic procedures, urologicprocedures, general surgical wounds and wounds in general. Compositionsof the invention may be delivered by, but not limited to, injection(e.g., via syringe), via irrigation fluid, as part of a bandage over awound, or in a topical application such as a solution, cream, gel or thelike.

In some embodiments of the invention, a complement factor B analogand/or vector of the invention is administered in combination with acomplement inhibiting factor, prior to, concurrently with, or afteradministration of the complement factor B analog and/or vector. Acomplement inhibiting factor includes, but is not limited to, a FactorH, a Factor H-like 1, an MCP, a DAF, or a soluble form of an MCP.

In some embodiments of the invention, a complement factor B analog orvector of the invention is administered in combination with ananti-angiogenic factor. Anti-angiogenic factors include, but is notlimited to, endostatin, a VEGF binding molecule, PEDF, T2-TrpRS (e.g.,see U.S. Pat. No. 7,273,844), sFLT (e.g., see Kong et al. Hum Gene Ther(1998) 9:823-833), aflibercept (VEGF Trap), VEGF Trap-eye, kininostatin,ranibizumab and bevacizumab.

In some embodiments, a complement factor B analog and/or vector of theinvention is administered in combination with LUCENTIS® (ranibizumab),AVASTIN® (bevacizumab), VEGF Trap-eye, aflibercept or a molecule(s) thatbinds VEGF and/or that inhibits angiogenesis. LUCENTIS® is used to treatwet AMD. Some embodiments of the invention can also be used to treat wetAMD. Therefore, the present invention provides methods and compositionsfor treating wet AMD comprising administering, separately or together, acomposition of the invention in combination with LUCENTIS®(ranibizumab), AVASTIN® (Bevacizumab) VEGF Trap-eye, aflibercept and/ora molecule(s) that binds VEGF and/or that inhibits angiogenesis.Additionally, intraocular inflammation is one of the most common adversereactions reported after administration of LUCENTIS®, e.g., see the“Full Prescribing Information” for LUCENTIS®. The present inventionprovides a method for inhibiting or reducing intraocular inflammation(e.g., resulting from the administration of LUCENTIS®) comprisingadministering a molecule or composition of the invention prior to, atthe same time, and/or after the administration of LUCENTIS®, VEGFTrap-eye or aflibercept.

In some embodiments of the invention, a complement factor B analog orvector of the invention is administered in combination with anothercompound(s), such as a compound that inhibits T-cell activation,B-cells, TNF, interleukin-1 (e.g., interleukin-1b), interleukin-6 and/orinterferon-gamma. A complement factor B analog or vector of theinvention can also be administered in combination with a compound(s)that inhibits complement activity, e.g., alternative complementactivity. Compounds that can be used and which inhibit TNF include, butare not limited to, compounds that bind TNF, such as antibodies (e.g.,Infliximab (REMICADE®), Golimumab (SIMPONI®) and Adalimumab (HUMIRA®))or soluble receptors that bind TNF such as Etanercept (ENBREL®). Othercompounds that can be used and which inhibit T-cell activation include,but are not limited to, compounds that bind B7 such as abatacept(ORENCIA®). Also compounds which downregulate B-cells can be usedincluding, but not limited to, compounds that bind CD20 such asRituximab (RITUXAN® and MABTHERA®).

Complement pathways contributing to and/or causing a disease can bemodulated, regulated, inhibited and/or activated using various methodsand/or complement factor B protein analogs that are part of the presentinvention.

Compositions, Formulations and Preparations

Some embodiments of the invention provide compositions, e.g.,pharmaceutical compositions containing a complement factor B analog ofthe invention, such as for therapeutic uses. In some embodiments, apharmaceutical composition comprises a complement factor B analogcomprising amino acids 26-764 of SEQ ID NO:2, amino acids 26-764 of SEQID NO:3, amino acids 26-990 of SEQ ID NO:22 or amino acids 26-990 of SEQID NO:23, for example, hfB3-2925 (SEQ ID NO:2), hfB3-2925-740N (SEQ IDNO:3), hfB3-2925-Fc (SEQ ID NO:22) or hfB3-2925-740N-Fc (SEQ ID NO:23).In some embodiments, a pharmaceutical composition comprises a complementfactor B analog consisting of amino acids 26-764 of SEQ ID NO:2, aminoacids 26-764 of SEQ ID NO:3, amino acids 26-990 of SEQ ID NO:22 or aminoacids 26-990 of SEQ ID NO:23. Examples of pharmaceutical compositionsand formulations that can be used with the complement factor B analogsof the invention are described in PCT Publication No. WO08/106644 andU.S. Patent Publication No. US20100120665.

Some embodiments of the invention include pharmaceutical preparationscomprising a complement factor B protein analog of the invention, anucleic acid of the invention, a viral vector of the invention or anycombination thereof.

Formulations (e.g., for injection) are generally, but not necessarily,biocompatible solutions of the active ingredient, e.g., comprisingHank's solution or Ringer's solution. Formulations for transdermal ortransmucosal administration generally include, but are not limited,penetrants such as fusidic acid or bile salts in combination withdetergents or surface-active agents. In some embodiments, formulationscan be manufactured as aerosols, suppositories, or patches. In someembodiments, oral administration may not be favored for protein orpeptide active ingredients; however, this type of composition may besuitably formulated, e.g., in an enteric coated form, in a depot, in acapsule and so on, so as to be protected from the digestive enzymes, sothat oral administration can also be employed. Some formulations of theinvention comprise balanced salt solution (Alcon Laboratories, Inc.,Fort Worth, Tex.) or balanced salt solution plus (Alcon Laboratories,Inc.). In some embodiments, a formulation comprises one or more of thefollowing: citrate, NaCl (e.g., 0.64%), potassium chloride (KCl) (e.g.,0.075%), calcium chloride dihydrate (CaCl₂.2H₂O) (e.g., 0.048%),magnesium chloride hexahydrate (MgCl₂.6H₂O) (e.g., 0.03%), sodiumacetate trihydrate (CH₃CO₂Na.3H₂O) (e.g., 0.39%), sodium citratedihydrate (C₆H₅O₇Na₃.2H₂O) (e.g., 0.17%), sucrose and sodium hydroxideand/or hydrochloric acid (to adjust pH) and water. The preceding listincludes some molecules that are listed as particular hydrates, e.g.,dihydrate, trihydrate, hexahydrate, etc. It is understood that varioushydrates of these compounds can be used in the present invention and theinvention is not limited to these particular hydrate forms of the listedmolecules. In some embodiments, a formulation comprises one or more ofthe following: NaCl, monobasic phosphate monohydrate, dibasic sodiumphosphate heptahydrate and hydrochloric acid and/or sodium hydroxide toadjust pH and water. In some embodiments, a pharmaceutical compositioncomprises at least one ingredient selected from the group consisting ofhistidine, MgCl₂, trehalose, a polysorbate, polysorbate 20, NaCl,sucrose, arginine and proline. In some embodiments, a formulationcomprises one or more of the following: histidine (e.g., about 10 mM);α, α-trehalose dehydrate (e.g., about 10% or about 50 mM); MgCl₂ (e.g.,about 10 mM); a polysorbate such as polysorbate 20 (e.g., about 0.01%);and NaCl (e.g., about 0.1%). In some embodiments, a formulation maycomprise one or more of the following: sucrose, arginine or proline. Insome embodiments, a formulation comprises or consists of a molecule(s)of the present invention, 10 mM histidine, 10 mM MgCl₂, 50 mM trehaloseand 0.01% polysorbate 20. In some embodiments, a formulation comprisesor consists of a molecule(s) of the present invention, 1.0% NaCl and 10mM MgCl₂. In some embodiments, a formulation comprises or consists of amolecule(s) of the present invention, and a balanced salt solutionenriched with bicarbonate, dextrose, and glutathione, such as BSS PLUS®.In some embodiments, a formulation does not comprise trehalose. In someembodiments, a formulation or composition is at a pH of about 5.5. Insome embodiments, a formulation or composition is at a pH of betweenfrom about 5.0 to 9.0, about 5.0 to 5.5, about 5.3 to 5.7, about 5.5 to6.0, about 5.8 to 6.2, about 6.0 to 6.5, about 6.3 to 6.7, about 6.5 to7.0, about 6.8 to 7.2, about 7.0 to 7.5, about 7.3 to 7.7, about 7.5 to8.0, about 7.8 to 8.2, about 8.0 to 8.5, about 8.3 to 8.7 and about 8.5to 9.0, whatever is suitable to retain the biological activity andstability of the active ingredient(s).

Some formulations of the invention can be manufactured as aerosols,suppositories, eye drops or patches.

Examples of suitable formulations and formulatory methods for a desiredmode of administration may be found in Remington's PharmaceuticalSciences, latest edition, Mack Publishing Co., Easton, Pa. and in U.S.Pat. No. 7,208,577.

In some embodiments, a composition for use in vivo contains a “carrier”or a “pharmaceutically acceptable carrier”. The term “carrier” refers toa diluent, adjuvant, excipient, or vehicle with which the vector ofinterest is administered. The term “carrier’ includes, but is notlimited to, either solid or liquid material, which may be inorganic ororganic and of synthetic or natural origin, with which an activecomponent(s) of the composition is mixed or formulated to facilitateadministration to a subject.

In general, a suitable oil(s), saline, aqueous dextrose (glucose), andrelated sugar solutions and glycols such as propylene glycol orpolyethylene glycols are typically suitable carriers for parenteralsolutions. In some embodiments, solutions for parenteral administrationcontain a water soluble salt of the active ingredient, suitablestabilizing agents, and if desirable or necessary, buffer substances.Antioxidizing agents such as sodium bisulfite, sodium sulfite, orascorbic acid, either alone or combined, can be used as stabilizingagents. Also used are citric acid and its salts and sodium EDTA. Inaddition, parenteral solutions can contain preservatives, such asbenzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.

Carriers can include carbohydrates such as trehalose, mannitol,glutathione, xylitol, sucrose, lactose, and sorbitol. Other ingredientsfor use in formulations may include, for example, DPPC(1,2-Didecanoyl-sn-glycero-3-phosphocholine), DOPE(1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), DSPC(1,2-Distearoyl-sn-glycero-3-phosphocholinez1,2-Distearoyl-sn-glycero-3-phosphocholine) and DOPC(1,2-Dioleoyl-sn-glycero-3-phosphocholine). Natural or syntheticsurfactants may be used. Polyethylene glycol may be used (even apartfrom its use in derivatizing a protein). Dextrans, such as cyclodextran,may be used. In some embodiments, cyclodextrin, tertiary amines and/orbeta-cyclodextrin may be used. Bile salts and other related enhancersmay be used. Cellulose and cellulose derivatives may be used. Aminoacids may be used, such as use in a buffer formulation. Also, the use ofliposomes, microcapsules or microspheres, inclusion complexes, or othertypes of carriers is contemplated.

Suitable pharmaceutical excipients include, but are not limited to,starch, glucose, lactose, sucrose, gelatin, antibiotics, preservatives,malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. A composition, ifdesired, can also contain wetting and/or emulsifying agents, and/or pHbuffering agents. Where necessary, a composition may also include asolubilizing agent and/or a local anesthetic such as lignocaine to easepain at the site of the injection.

Also contemplated herein is pulmonary delivery of an agent or protein(or derivative thereof) of the present invention. In some embodiments, acomplement factor B analog(s) is delivered to the lungs of a mammalwhile inhaling and can mostly remain in the lungs or in some embodimentstraverses across the lung epithelial lining to the blood stream. (e.g.,see Adjei et al., Pharmaceutical Research 7:565-569 (1990); Adjei etal., International Journal of Pharmaceutics 63:135-144 (1990); Braquetet al., Journal of Cardiovascular Pharmacology 13(suppl. 5):s.143-146(1989); Hubbard et al., Annals of Internal Medicine 3:206-212 (1989);Smith et al., J. Clin. Invest. 84:1145-1146 (1989); Oswein et al.,Proceedings of Symposium on Respiratory Drug Delivery II, Keystone,Colo., March, 1990; Debs et al., The Journal of Immunology 140:3482-3488(1988) and Platz et al., U.S. Pat. No. 5,284,656). Contemplated for usein the practice of this invention are a wide range of mechanical devicesdesigned for pulmonary delivery of therapeutic products, including butnot limited to nebulizers, metered dose inhalers, and powder inhalers.Some specific examples of commercially available devices suitable forthe practice of some embodiments of the invention are the ULTRAVENT™nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the ACORNII® nebulizer, manufactured by Marquest Medical Products, Englewood,Colo.; the VENTOLIN metered dose inhaler, manufactured by Glaxo Inc.,Research Triangle Park, N.C.; and the SPINHALER powder inhaler,manufactured by Fisons Corp., Bedford, Mass.

In some embodiments, a protein is prepared in particulate form. In someembodiments, this particulate form has an average particle size of lessthan 10 μm (or microns), most preferably 0.5 to 5 μm, for delivery tothe distal lung.

Formulations suitable for use with a nebulizer (e.g., jet or ultrasonic)will typically comprise a complement factor B analog dissolved in water,in some embodiments, at a concentration of about 0.1 to about 25 mg ofbiologically active protein per mL of solution. A formulation may alsoinclude a buffer and/or a simple sugar (e.g., for protein stabilizationand regulation of osmotic pressure). A nebulizer formulation may alsocontain a surfactant, to reduce or prevent surface induced aggregationof a protein(s) caused by atomization of the solution in forming theaerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing a complement factor B analogof the invention suspended in a propellant, e.g., with the aid of asurfactant. A propellant may be any conventional material employed forthis purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude sorbitan trioleate and soya lecithin. Oleic acid may also beuseful as a surfactant. In some embodiments, formulations for dispensingfrom a powder inhaler device will comprise a finely divided dry powdercontaining a complement factor B analog of the invention and may alsoinclude a bulking agent, such as lactose, sorbitol, sucrose, mannitol,trehalose, or xylitol in amounts which facilitate dispersal of thepowder from the device, e.g., 50 to 90% by weight of the formulation.

Administration and Delivery

It is understood that when introduction or administration of a nucleicacid encoding a complement factor B protein analog is discussed, thatthe invention also contemplates the introduction or administration ofthe complement factor B protein analog itself. It is understood thatwhen introduction of a complement factor B analog is discussed, that theinvention also contemplates the introduction of a nucleic acid encodingthe complement factor B protein analog.

In some embodiments, complement factor B analogs or compositions of theinvention can be administered locally or systemically. Useful routes ofadministration are described herein and known in the art. Methods ofintroduction or administration include, but are not limited to,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, intratracheal, topical, inhaled, transdermal, rectal,parenteral routes, epidural, intracranial, into the brain,intraventricular, subdural, intraarticular, intrathecal, intracardiac,intracoronary, intravitreal, subretinal, intraanterior chamber of theeye, particular, locally on the cornea, subconjunctival, subtenoninjection, by applying eyedrops, oral routes, via balloon catheter, viastent or any combinations thereof. In some embodiments, a composition orcomplement factor B analog of the invention is administered to a drusen,e.g., by injecting directly into a drusen. Systemic administration maybe, but is not limited to, by intravenous or intra-arterial injection orby transmucosal, subcutaneous and/or transdermal delivery. In someembodiments, a composition of the invention may be initially directed toa site other than a diseased site. For example regarding AHR whichoccurs in the lungs of an animal, an intraperitoneal injection of aprotein, vector or nucleic acid of the invention may result in a changein AHR in the lungs, e.g., see Park et al., American Journal ofRespiratory and Critical Care Medicine 169:726-732, (2004). In someembodiments, a dosage level and/or mode of administration of acomposition may depend on the nature of the composition, the nature of acondition(s) to be treated, and/or a history of an individual patient.In some embodiments, cells expressing a complement factor B analog ofthe invention are administered. These cells can be a cell line,xenogeneic, allogeneic or autologous.

In some embodiments, e.g., comprising administration to the eye, acomplement factor B protein analog or vector of the invention isadministered about once every week, month, 2 months, 3 months, 6 months,9 months, year, 18 months, 2 years, 30 months, 3 years, 5 years, 10years or as needed. In some embodiments, e.g., comprising administrationto the eye, a molecule or vector of the invention is administered fromabout every 1 to 4 weeks, about every 4 to 8 weeks, about every 1 to 4months, about every 3 to 6 months, about every 4 to 8 months, aboutevery 6 to 12 months, about every 9 to 15 months, about every 12 to 18months, about every 15 to 21 months, about every 18 to 24 months, aboutevery 1 to 2 years, about every 1.5 to 3 years, about every 2 to 4years, about every 3 to 5 years, about every 5 to 7 years, about every 7to 10 years or about every 10 to 20 years. It is expected thatadministration of a vector coding for a complement factor B proteinanalog would be less frequent than administration of the complementfactor B protein analog. In some embodiments of the invention, apharmaceutical preparation comprises a vector encoding a complementfactor B analog of the invention and the pharmaceutical preparation isadministered only once to the patient.

In some embodiments, e.g., comprising administration to the eye, avector coding for a complement factor B analog is administered 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more times to a patient in their lifetime. Insome embodiments, e.g., comprising administration to the eye, alentiviral vector of the invention is administered 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more times to a patient in their lifetime.

In some embodiments, a complement factor B protein analog of theinvention is administered by intravitreal injection to a human eye. Insome embodiments, about 15 μg to about 5 mg; about 15 μg to about 500μg; about 100 μg to about 900 μg; about 300 μg to about 700 μg; about500 μg to about 1 mg; about 1 mg to about 5 mg; about 1 mg; or about 500μg of a complement factor B protein analog is administered byintravitreal injection to a human eye.

In some embodiments, a complement factor B protein analog of theinvention is administered by subretinal injection or intravitrealinjection of a lentiviral or adeno associated viral (AAV) vector. Insome embodiments, about 5×10⁶ to about 5×10⁸; about 5×10⁶ to about5×10⁷; about 5×10⁷ to about 5×10⁸; about 1×10⁷ to about 1×10⁸; about3×10⁷ to about 5×10⁷; about 2.5×10⁷; about 5×10⁷; about 7.5×10⁷; orabout 1×10⁸ transducing units of a lentiviral vector is administered bysubretinal injection. In some embodiments, about 5×10⁸ to about 1×10⁹;about 5×10⁸ to about 7.5×10⁸; about 7.5×10⁸ to about 1×10⁹; about 6×10⁸to about 9×10⁸; about 7×10⁸ to about 8×10⁸; about 5×10⁸; about 6×10⁸;about 7×10⁸; about 8×10⁸; about 9×10⁸; or about 1×10⁹ transducing unitsof an AAV vector is administered by subretinal injection.

In some embodiments, about 5×10⁸ to about 1×10¹⁰; about 5×10⁸ to about5×10⁹; about 5×10⁸ to about 2×10⁹; about 2×10⁹ to about 5×10⁹; about5×10⁹ to about 1×10¹⁰; about 5×10⁸ to about 1×10⁹; about 1×10⁹ to about3×10⁹; about 3×10⁹ to about 6×10⁹; about 6×10⁹ to about 1×10¹⁰; or about1×10⁹ to about 1×10¹⁰ transducing units of an AAV vector is administeredby intravitreal injection.

In some embodiments, about 50 μl to about 100 μl, about 50 μl to about75 μl, about 75 μl to about 100 μl, about 60 μl to about 90 μl, about 70μl to about 80 μl, about 50 μl; about 60 μl; about 70 μl; about 80 μl;about 90 μl; or about 100 μl of a complement factor B protein analog ora vector encoding a complement factor B protein analog is injectedsubretinally. In some embodiments, about 50 μl to about 1 ml, about 50μl to about 500 μl, about 500 μl to about 1 ml, about 250 μl to about750 μl, about 250 μl to about 500 μl, about 500 μl to about 750 μl,about 400 μl to about 600 μl, or about 750 μl to about 1 ml of acomplement factor B protein analog or a vector encoding a complementfactor B protein analog is injected intravitreally.

In some embodiments, an anti-inflammatory may be delivered incombination with a complement factor B protein analog (e.g., hfB3-292Sor hfB3-292S-740N), vector or nucleic acid of the invention. Ananti-inflammatory may be delivered prior to, concurrently with, and/orafter administration of a molecule or vector of the invention. In someembodiments, an anti-inflammatory is administered in the same solutionand/or same syringe as a complement factor B protein analog, nucleicacid or vector of the invention. In some embodiments, a complementfactor B protein analog or vector of the invention and ananti-inflammatory are co-administered to the eye, e.g., as describedherein.

Many anti-inflammatory drugs are known in the art and include, but arenot limited to, dexamethasone, dexamethasone sodium metasulfobenzoate,dexamethasone sodium phosphate, fluorometholone, bromfenac, pranoprofen,RESTASIS™, cyclosporine ophthalmic emulsion, naproxen, glucocorticoids,ketorolac, ibuprofen, tolmetin, non-steroidal anti-inflammatory drugs,steroidal anti-inflammatory drugs, diclofenac, flurbiprofen,indomethacin, and suprofen.

Some embodiments of the invention include administration of both acomplement factor B protein analog and a vector encoding it. Acomplement factor B protein analog of the invention may be deliveredprior to, concurrently with, and/or after administration of a vector ofthe invention. In some embodiments, a complement factor B protein analogof the invention is administered in the same solution and/or samesyringe as a vector of the invention. In some embodiments, a complementfactor B protein analog of the invention and a vector of the inventionare co-administered to the eye, e.g., as described herein.

Additionally, a complement factor B analog or a nucleic acid encoding itcan be delivered or administered to an animal via a cell, e.g., as celltherapy. For example, this can be accomplished by administering ordelivering a cell(s) expressing a complement factor B analog(s). In someembodiments, a complement factor B analog(s) is expressed from the cellvia a regulatable, inducible and/or repressible promoter. In someembodiments, encapsulated cells that express a complement factor Banalog(s) are delivered to an animal, e.g., see PCT Publication No.WO07078922 related to encapsulated cells. In some embodiments, cells areadministered locally (e.g., in a joint, intravitreal, intraretinal,intracranially etc.) or systemically (e.g., i.v.).

Cells to be administered to an animal can be autologous, allogeneic orxenogeneic. In some embodiments, autologous cells are manipulated exvivo to cause them to produce a complement factor B protein analog ofthe invention and, in some embodiments, the cells are introduced back tothe animal. Transferring a nucleic acid comprised of a coding region tocells ex vivo can be by any method, such as, electroporation,microinjection, cell fusion, chromosome-mediated gene transfer,microcell-mediated gene transfer, spheroplast fusion, lipofection,microparticle bombardment, calcium phosphate mediated transfection,viral infection and so on. Optionally, a selectable marker also can beintroduced into the cells. If a selectable marker is utilized, the cellscan be then placed under selection, e.g., to enhance expression and/orto isolate those cells that express the transferred coding region (see,e.g., Loeffler & Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al.,Meth. Enzymol. 217:618-644 (1993); and Cline, Pharmac. Ther. 29:69-92(1985)).

Recombinant cells (e.g., autologous or allogeneic cells transduced invitro) can be delivered to a patient by various methods known in theart. For example, cells can be encapsulated prior to administration, asknown in the art. In some embodiments, when encapsulated, the cells arenot autologous. In some embodiments, recombinant blood cells (e.g.,hematopoietic stem and/or progenitor cells) are administeredintravenously. In some embodiments, eye cells and/or pluripotentialcells can be injected directly into the eye. The amount of cells neededdepends on the desired effect, the animal's state, etc.

In some embodiments of the invention, a gene delivery system can resultin transduction and/or stable integration of a gene or coding region fora complement factor B analog into a target cell. In some embodiments,target cells are mammalian cells such as primate cells, and human cells.In some embodiments, target cells are cells of the eye, such as retinalpigment epithelial cells, retinal cells, or pluripotential cells. Targetcells can be in vitro, ex vivo or in vivo. In some embodiments, a targetcell is a stem cell. Stem cells include, but are not limited to,pluripotent stem cells, totipotent stem cells, hematopoietic stem cells,cancer stem cells and embryonic stem cells. In some embodiments,pluripotential cells contemplated herein are not those for propagating aliving entity from a zygote or blastomere. The instant invention alsocontemplates the use of a partially undifferentiated cell forimplantation into the eye of a patient in need of treatment, e.g., toregenerate cells of the eye.

Transgenic Animals

Some embodiments of the invention provide a transgenic animal (e.g.,nonhuman) expressing a complement factor B analog of the invention.Methods for making a transgenic animal are known in the art. In someembodiment, a transgenic animal (such as a mouse) will also comprise amutation, deletion or disruption in the Fas gene, e.g., see Macmickinget al. Cell. 81:641-650 (1995).

EXAMPLES

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseexamples but rather should be construed to encompass any and allvariations which become evident as a result of the teachings providedherein.

Whereas, particular embodiments of the invention have been describedherein for purposes of description, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

Example 1. Generation of hfB3 Expression Construct

A plasmid was designed to include the coding sequence for human hfB3with an IRES-Neo selectable marker (hfB3-IRES-Neo). The plasmid wassynthesized by GENEART AG (Regensburg, Germany, plasmid), a fee forservice contract organization. An Nhe I restriction site wasincorporated into both the 5′ and 3′ ends of the coding sequence. ThehfB3 nucleic acid coding sequence was codon optimized for optimalexpression in mammalian cells.

The gene expression plasmid, pCI (Promega, Madison, Wis.), was modified.First, the BGH (Bovine Growth Hormone) polyA was removed from pCI andreplaced with a synthetic polyA. Next, the hfB3 coding sequence with aselectable marker (hfB3-IRES-Neo) was cut out by Nhe I from a plasmidand cloned into the Sal I site of the modified pCI as a blunt-endligation to create an hfB3 expression construct. The plasmid wassequenced in its entirety to confirm the sequence integrity of theconstruct (SEQ ID NO:5).

Example 2. Generation of hfB3-292S Expression Construct

A further modification was introduced to hfB3 protein. Human wild typefactor B protein and hfB3 protein have 23 cysteine amino acids (C),suggesting there is at least one unpaired free cysteine present in theprotein. Disulfide bond mapping suggested the free C in biologicallyactive hfB3 is located at the amino acid 292. The C at 292 is highlyconserved in factor B protein among different species (Table 1, above).In this Example, this C at 292 is changed to serine (S), generatinghfB3-2925.

To create the hfB3-2925 expression construct, site-specific mutation wasintroduced into the hfB3 expression construct (SEQ ID NO:5).

The hfB3 expression construct was used as the template to make the sitemutation changing the C at position 292 to S using Stratagene'sSite-Directed Mutagenesis Kit according to the manufacture'sinstructions, creating the hfB3-2925 expression construct. Two primerswere used: Forward primer 5′-CACCGGCGCCAAGAAGAGCCTGGTCAACCTGATC-3′ (SEQID NO:6) and Reverse primer 5′-GATCAGGTTGACCAGGCTCTTCTTGGCGCCGGTG-3′(SEQ ID NO:7). The underlined nucleotides indicate the mutated aminoacid from C to S. The hfB3-2925 expression cassette includes, from 5′ to3′, a CMV promoter, a chimeric intron, a codon optimized coding sequencefor hfB3-2925, an IRES-Neo selectable marker, and a synthetic polyA(FIG. 2). The entire construct was sequenced to confirm the mutation andthe integrity of the construct (SEQ ID NO:8). The expected amino acidsequence for hfB3-2925 is shown in SEQ ID NO:2.

Example 3. Generation of Stable hfB3 and hfB3-2925 Expression Cell Lines

Stable cell lines expressing hfB3 or hfB3-2925 protein were generated bytransfecting 293 FreeStyle cells (Invitrogen, Cat. No. R79007) with thehfB3 or hfB3-2925 expression construct. Transfection of plasmid DNA intothe 293 FreeStyle cells was mediated by PEI (Polyethylenimine, Sigma,Cat. No. 23966)-based transfection. A PEI solution was prepared insterile water at a final concentration of 1 mg/mL. The pH was adjustedto 7.0 with 5 N HCl. The solution was sterilized using a 0.22 μm filter.Aliquots of the PEI were stored frozen at −80° C. until use.

The transfection protocol was as follows:

One day prior to transfection, the cells were seeded at 1×10⁶ cells/mLin serum-free 293F Expression Medium (Invitrogen, Cat. No. 12338-018).

The next day, the cells were washed with basal RPMI1640 medium(Invitrogen, Cat. No. 22400-089) supplemented only with HT Supplement(Cat. No. 11067-030, Invitrogen), resuspended in the same medium at2×10⁶ cells/mL and dispensed into a new 6-well plate with 1 mL in eachwell.

Stock solutions of DNA and PEI were prepared in sterile 150 mM NaCl asfollows: 2.5 μg hfB3 or hfB3-2925 expression construct DNA (in 2.5 μL)was diluted into 47.5 μL of 150 mM NaCl and mixed with pipetting (DNAsolution). Ten microliters (10 μL) of PEI solution was diluted into 40μL of 150 mM NaCl, followed by a gentle vortex (PEI solution). The DNAand PEI solutions were incubated at room temperature for 5 minutes. ThePEI solution was then added to the DNA solution and the mixture wasallowed to incubate at room temperature for an additional 10 minutes andthen the DNA/PEI mixture was added to the cells in the 6-well plate andthe cells were incubated with agitation (200 RPM) for 5 hours at 37° C.in an incubator with 8% CO₂ and 85% humidity. After 5 hours, 1.1 mL ofcomplete 293F Expression Medium (no additives) was added to each of thewells and the incubation was continued for 72 hours.

The cells were then harvested, washed once with the 293F ExpressionMedium, and placed into fresh 293F Expression Medium containing 300μg/mL G418 (Teknova). As a negative control, an equal number ofun-transfected 293F naive cells were cultured in the sameG418-containing medium. The cells were under G418 selection forapproximately 3 weeks. By which time, the un-transfected cells in theG418-containing medium were dead. The transfected cells were passed overa FICOL gradient (Sigma) to remove the dead or dying cells from theG418-resistant live population. The G418-resistant live population wasfurther expanded over a period of about 2 weeks, during which the cellswere spun down every 2-3 days and resuspended in fresh 293F ExpressionMedium containing 300 μg/mL G418.

Example 4. Production of hfB3 and hfB3-292S

The G418-resistant hfB3 or hfB3-2925 producing cells were seeded at adensity of 2×10⁶ cells/mL in the 293F Expression Medium either in 6-wellplates with 2 mL culture in each well, in 500 mL spinner flasks with 100mL culture in each flask, or in 3,000 mL spinner flasks with 1,000 mLculture in each flask. The cells were incubated for 72 hours withshaking at 100 rpm on an orbital shaker in a 37° C. incubator with 8%CO₂ and 80% humidity.

The cell culture medium supernatant containing hfB3 protein or hfB3-292Sprotein was then harvested and centrifuged at 2,000 rpm for 10 minutesto clear cell debris after which the culture medium was filtered througha 0.22 μm filter.

Example 5. Quantitation of hfB3 and hfB3-292S Proteins

An Electrochemiluminescent assay (ECL) was developed for quantitation ofhfB3 and hfB3-292S with human wild type factor B as standard. The assaywas a sandwich immunoassay based on BioVeris's ECL Technology. Briefly,the ECL assay is formatted as a 96-well plate sandwich, one-step, and nowash assay. The quality control samples (purified factor B from humanplasma, Quidel, Cat. No. A408) and test samples were incubated with amaster mix reagent containing a biotinylated anti-hfB monoclonalantibody (anti-human factor B monoclonal antibody, R&D Systems, Cat. No.MAB2739), a BV-TAG plus-labeled anti-hfB polyclonal antibody (anti-humanfactor B polyclonal antibody, R&D Systems, Cat. No. AF2739), andstreptavidin-coated paramagnetic beads. The mixture was incubated for150 minutes. Following the incubation, a stop solution (Borate Buffer,250 mM, pH9.2 containing 500 mM sodium chloride and 1.6 mg/mL BSA) wasadded and then the plate was read on M1MR Analyzer. The estimateddynamic range for the assay was 9.0 to 950 ng/mL.

Example 6. Western Blot Analysis for hfB3 and hfB3-292S

Polyacrylamide electrophoresis of hfB3 protein and hfB3-2925 proteinunder denturing but non-reducing conditions (SDS-PAGE) was performed bymixing samples of hfB3 protein or hfB3-2925 protein with non-reducingprotein sample buffer (Pierce). Human factor B protein purified fromplasma (100 ng per sample, Quidel) was used as a positive control. Eachget also contained a well with pre-stained protein molecular weightmarkers (15 μL/lane) (Invitrogen). The samples and the markers wereheated at 95° C. for 5 minutes in non-reducing protein sample buffer.The samples were loaded onto a 7.5% Tris-HCL Precast mini gel (Bio-Rad).The gel was run (10×SDS/Tris/Glycine Running Buffer, Bio-Rad) at 75 Vfor 15 minutes or until the dye front passed through the stacking gelinto the resolving gel. Once the dye front entered the resolving gel,the voltage was increased to 100 V and electrophoresis contintued untilthe dye front ran off the gel.

Western blot analysis was performed by washing and equilibrating the gelin transfer buffer (10× Tris/Glycine Transfer Buffer, Bio-Rad) for 20minutes while rocking gently. A nitrocellulose membrane (Bio-Rad) andblotting paper were also equilibrated in the transfer buffer. Proteinsseparated by SDS-PAGE were transferred electrophoretically onto anitrocellulose using a Trans Blot Semi-Dry Transfer Cell (Bio-Rad) (20Vfor 45 minutes). Once the transfer was complete, the membrane wasblocked with a 1× casein solution (Vector Laboratories) for at least anhour at room temperature with gentle agitation on a rocker. The membranewas probed with a primary antibody (monoclonal antibody against humanfactor B, R&D Systems, Cat. No. MAB2739) diluted to 1:10,000 in 1×casein solution at room temperature for 1 hour with gentle agitation andwashed in 10 mL of 1× casein solution 3 times for 5 minutes each at roomtemperature with gentle agitation on a rocker. The membrane wasincubated with a biotinylated goat anti-mouse IgG (secondary antibody,R&D Systems, Cat. No. BAF007), diluted to 1:20,000 in 1× caseinsolution, for 1 hour at room temperature with gentle agitation on arocker and washed in 10 mL of 1× casein solution 3 times for 5 minuteseach at room temperature with gentle agitation. The membrane wasincubated in Vectastain ABC-AmP reagent (Vector Laboratories) in 20 mLof 1× casein solution for 45 minutes containing 40 μL of Reagent A and40 μL of Reagent B. The membrane was washed in 10 mL of 1× caseinsolution 3 times for 5 minutes each at room temperature with gentleagitation.

To aquire the chemiluminescent signal from the Western blots, themembranes were equilibrated in 20 mL of 0.1 M Tris pH 9.5 for 5 minuteswithout agitation. Excess buffer was removed from the membrane byholding the membrane vertically and touching the edge of the membrane toa Kimwipe. The target side of the membrane was placed facing up in a newcontainer. Duolox Substrate (7 mL, Vector Laboratories) was placeddirectly onto the target side of the membrane which was incubated for 5minutes in the dark. Excess Duolox was removed from the membrane byholding the membrane vertically and touching the edge of the membrane toa Kimwipe. The membrane was washed by submerging it in 20 mL of 0.1 MTris pH 9.5 for 5 minutes with agitation in the dark. Excess buffer wasremoved from the membrane by holding the membrane vertically andtouching the edge of the membrane to a Kimwipe. The membrane was placedin a folded plastic wrap sheet and exposed to Kodak BioMax MS X-ray filmin a film cassette for 1 to 5 minutes. The film was placed in KodakDeveloper solution (dilute 26 mL of the Developer solution into 92 mL ofddH₂O) for 1 minute. The film was removed from the Developer solutionand placed in Kodak Fixer solution (dilute 26 mL of the Fixer solutioninto 92 mL of ddH₂O) for 1 minute. Finally, the film was rinsed with tapwater and allowed to dry at room temperature.

As shown in FIG. 3, G418-resistant hfB3 and hfB3-292S producing cellsproduced, in the cell culture medium, hfB3 protein (Lane 3) andhfB3-292S protein (Lane 4) at the appropriate size (approximately 100KDa). These proteins migrated at approximately the same rate as the wildtype human factor B prufied from human plasma (Lane 2). No visible bandwas detected in the un-transfected cell culture medium (negativecontrol) indicating the specificity of the monoclonal anti-human factorB antibody (Lane 1). Interestingly, presumed aggregates at approximately200-260 KDa were readily detected in hfB3 samples (lane 3), while noaggregates were detected in the hfB3-292S samples (lane 4) producedunder the same experimental conditions. Aggregates could be caused bymisfolded populations of hfB3 in the cell culture medium. When proteinsare misfolded, they expose hydrophobic regions that are prone to theformation of aggregates through hydrophobic-hydrophobic interactions.These data suggest that in the hfB3-292S protein preparation, misfoldingwas either eliminated or significantly reduced as compared to hfB3protein.

Example 7. Alternative Complement Pathway Hemolytic Activity Assay

Human alternative complement pathway activity can be measured using ahemolytic assay as described in this Example.

One milliliter (1 mL) of rabbit erythrocytes (rRBCs) (Lampire BiologicalLaboratory, Cat. No. 7246408) suspension was washed with freshly madecold Mg²⁺-EGTA buffer. The erythrocytes were transferred to a 50 mLconical centrifuge tube, 30 mL of the Mg²⁺-EGTA buffer was added and thecells were mixed gently. The rRBCs were pelleted in a Beckman Allegra6KR centrifuge at 1,200 rpm at 4° C. without brake for 5 minutes andresuspended in the Mg²⁺-EGTA buffer. This wash step was repeated twice.The rRBCs were resuspended in 2 mL of ice-cold Mg²⁺-EGTA buffer and acell count was obtained using a hemocytometer.

The hemolytic activity reaction mixture was set up in V-bottom shaped96-well plates placed on ice. To determine if hfB3 protein or hfB3-292Sprotein can compete with the wild type human factor B protein andinhibit its hemolytic activity, a competition assay was set up in atotal volume of 40 μL including 500 ng of wild type human factor B,increasing amounts of hfB3 protein or hfB3-292S protein, and GVB⁺⁺buffer (Sigma, Cat. No. G6415). Fifty microliters (50 μL) of factor Bdepleted human serum diluted 25-fold with Mg²⁺-EGTA buffer was added toeach well, followed by 10 μL of Mg²⁺-EGTA washed 5×10⁷rRBCs. Afteradding the rRBCs, each sample was gently mixed in the 96 well plateusing a multi-channel pipette.

The 96-well plate was placed in a glass tray with a layer of 37° C.water submerging the bottom of the plate. The tray was then placed in a37° C. water bath with orbital shaking at 110 rpm for 40 minutes. Afterincubation, the plate was placed on ice, 150 μL of ice-cold 0.9% salinewas added to each well, and each reaction gently mixed by pipetting tostop the reaction. The 96-well plate was centrifuged at 2,000 rpm in anEppendorf 5810R centrifuge for 5 minutes (min) at 4° C. without brake topellet the rRBC at the bottom of the plate. The supernatant (180 μL) wasremoved from each well without disturbing the pellet and transferred tothe corresponding well of a new 96-well plate. The absorbance of eachsample was measured at 405 nm in a microplate reader.

Example 8. Biological Activity of hfB3 and hfB3-292S

The biological activity of hfB3 protein and hfB3-2925 protein wasexamined by measuring the alternative complement pathway mediatedhemolysis of rRBCs. The assay described in Example 7 was used to testthe potency of hfB3 protein or hfB3-2925 protein in inhibiting the humanalternative complement pathway. Each reaction was performed intriplicate. As shown in FIG. 4, the raw cell culture medium fromhfB3-2925 protein and hfB3 protein producing cells efficientlyinhibited/reduced the alternative complement pathway activity in adose-dependent manner. Although not wishing to be bound by theory, hfB3and hfB3-2925 protein may be inhibiting alternative complement pathwayactivity by competing against the wild type factor B protein and/or bysequestering C3b and/or complement factor D.

Example 9. Purification of hfB3 and hfB3-292S Proteins

Cell culture medium from a human 293 FreeStyle cell line transfected andstably expressing and secreting hfB3 protein or hfB3-2925 protein wasused as the starting material for purification of hfB3 protein orhfB3-2925 protein. Soluble secreted hfB3 or hfB3-2925 protein waspurified from the cell culture supernatant by a combination of anionexchange (AEX), hydrophobic interaction (HIC) and size exclusionchromatography (SEC) for capturing, intermediate purification andpolishing steps, respectively, using a GE AKTA Purifier. Twopurification schemes are described below. These two schemes differ inthat one uses one HIC chromatography step and the other uses two HICchromatography steps.

The cell culture supernatant containing hfB3 protein was diluted withdistilled water at 4:1 volume ratio of culture supernatant/water tolower the conductivity to ˜8 Milli Siemens per centimeter (mS/cm) andadjusted to pH 7.5 with 50 mM phosphate buffer. This material was loadeddirectly onto a pre-packed ion exchange column (POROS HQ 50, ABI) on anAKTA purifier using a P-960 pump at a flow rate of 30 mL per minute. Thecolumn was previously equilibrated with buffer A (50 mM phosphate buffer(PB), pH7.5, conductivity ˜8 mS/cm), at a linear flow rate of 600 mL/hr(˜1 Column Volume/min, CV/min). The effluent was monitored by UVdetection at 280 nm. After washing away unbound material, retainedmaterial was eluted with a non-linear gradient of buffer A and buffer B(50 mM PB and 1 M NaCl, pH 7.5) to sequentially raise the conductivityof the mobile phase stepwise from 8 mS/cm (0% of buffer B for 2 CV), to33 mS/cm (25% of buffer B for 8 CV) and finally to 105 mS/cm (100% ofbuffer B for 5 CV).

To further facilitate removal of host protein contaminants, anintermediate, hydrophobic interaction (HIC) chromatography step wasintroduced, which purifies and separates proteins based on differencesin their surface hydrophobicity. The major fractions (from the AEXchromatography step) containing hfB3 protein were pooled and adjusted tocontain approximately 1.4 M ammonium sulfate and 47 mM phosphate buffer,pH 7.5 (conductivity 216 mS/cm) (1.0 M ammonium sulfate and 33.7 mMphosphate buffer, pH 7.5, conductivity 169 mS/cm for hfB3-2925) byadding buffer C (1.5 M ammonium sulfate and 50 mM phosphate buffer, pH7.5) to the sample at approximately 30:1 volume ratio of the ammoniumsulfate/phosphate buffer vs. the sample. The pooled sample was filteredthrough a 0.2 μm filter and applied to a hydrophobic interaction column(HiTrap Phenyl HP, GE Healthcare) pre-equilibrated with 1.5 M ammoniumsulfate and 50 mM Phosphate buffer, pH 7.5 (buffer C) (1.0 M ammoniumsulfate and 33.7 mM phosphate buffer for hfB3-2925) at the flow rate of300 mL/hr (1 CV/min). The retained material was eluted by decreasing theammonium sulfate concentration in a non-linear fashion.

Alternatively, the intermediate HIC chromatography step can be replacedwith a two-steps HIC chromatography, e.g., to make the purificationprocess easier to scale-up. In this two-step HIC purification process,the majority of host cell proteins were separated from fB3 or fB3-292Sby the first step HIC chromatography, HIC negative selection, by bindingto HIC column (HiTrap Phenyl HP, GE Healthcare) at low salt condition(0.75 M ammonium sulfate and 25 mM phosphate buffer, pH 7.5 for fB3 and0.6 M ammonium sulfate and 20 mM phosphate buffer, pH 7.5, conductivity100 mS/cm for hfB3-292S). The flow-thru fraction from the HIC negativeselection step containing hfB3 or fB3-292S protein was then re-adjustedto contain approximately 1.5 M ammonium sulfate and 50 mM phosphatebuffer, pH 7.5 (conductivity 216 mS/cm) for fB3 or 1.0 M ammoniumsulfate and 33.7 mM phosphate buffer, pH 7.5, (conductivity 169 mS/cm)for hfB3-292S. For the second step HIC chromatography, the sample wasfiltered through a 0.2 μm filter and applied to a hydrophobicinteraction column (HiTrap Phenyl HP, GE Healthcare) pre-equilibratedwith 1.5 M ammonium sulfate and 50 mM Phosphate buffer, pH 7.5 for fB3and 1.0 M ammonium sulfate and 33.7 mM phosphate buffer for hfB3-292S atthe flow rate of 300 mL/hr (1 CV/min). The retained material was elutedby decreasing the ammonium sulfate concentration in a non-linear fashionto further separate fB3 or fB3-292S protein from the remaining host cellproteins.

The fractions containing biologically active hfB3 protein from the HICchromatography step were pooled and concentrated with a centrifugalfilter device (Millipore, Amicon Ultra, Cat. No. 901024, 10,000 MWCut-off). The concentrated sample was subjected to size exclusionchromatography on a Sephacryl 5300 26/60 HR column (maximum loadingvolume: 3% of CV, ˜10 mL), equilibrated in PBS buffer (4 mM phosphate,150 mM NaCl, pH 7.4, (GIBCO)). The elution of hfB3 protein was performedat a constant linear flow rate of 60 cm/hr using PBS, monitoring theeffluent by UV detection at 280 nm. The purified hfB3 protein was storedat −80° C. in aliquots.

This procedure permitted almost complete separation of hfB3 protein fromother contaminants. The purity of hfB3 protein after the three stepchromatography process was quite high, as indicated by the fact thatSDS-PAGE silver staining analysis of 0.2 μg of purified hfB3 proteinonly produced a single sharp band (FIG. 5, left panel). When hfB3protein purified by the above three step chromatography process wassubjected to a competition assay against human wild-type factor B in ahemolytic assay, it suppressed hemolytic activity of the humanalternative complement pathway, demonstrating that the purified hfB3protein was biologically active (FIG. 5, right panel). Surprisingly, twopopulations of hfB3 were detected by HIC, one was biologically active(designated as Peak I or active population) and the other had a muchreduced biological activity (designated as Peak II or less activepopulation) (FIG. 6). These two forms were readily detected byreverse-phase HPLC (RP-HPLC) in the un-processed cell culture medium ofstable hfB3 protein producing cells (FIGS. 7A and 7B).

Example 10. hfB3-292S Protein Producing Cell Line does not Produce thePeak II Population

The raw hfB3-292S protein cell culture medium containing a complementfactor B analog with the free cysteine substituted with serine at theposition 292 was subjected to RP-HPLC analysis with raw naïve 293FreeStyle cell culture medium as a negative control and raw hfB3 proteincell culture medium as a positive control (FIGS. 7A & 7B). The hfB3-292Sprotein cell culture medium did not produce any detectable Peak IIpopulation, whereas analysis of hfB3 protein cell culture medium showedthat 35% of hfB3 was in the less active, Peak II, population. (FIGS. 7A& B.)

Example 11. Generation and Characterization of hfB4 Expression Constructand hfB4 Protein

hfB4 protein was designed to change the aspartic acid, at amino acid 740in hfB3, to an asparagine. This change is thought to attenuate orinhibit the function of the serine protease function of this complementfactor B protein analog.

To create the hfB4 expression construct, site-specific mutation wasintroduced into the hfB3 expression construct.

The hfB3 expression construct (SEQ ID NO:5) described in Example 1 wasused as a template to make a mutation changing the aspartic acid (D) atposition 740 in SEQ ID NO:4 to asparagine (N) using Stratagene'sSite-Directed Mutagenesis Kit (Stratagene, Santa Clara, Calif.)according to the manufacture's instructions, creating the hfB4expression construct. Two primers were used: Forward primer5′-GTCCCCGCCCACGCCCGGAACTTCCACATCAACCTGTTCC-3′ (SEQ ID NO:15) andReverse primer 5′-GGAACAGGTTGATGTGGAAGTTCCGGGCGTGGGCGGGGAC-3′ (SEQ IDNO:16). The underlined nucleotides indicate the nucleotide change thatresults in the amino acid change from D to N. This hfB4 expressionconstruct includes, from 5′ to 3′, a CMV promoter, a chimeric intron, acodon optimized coding sequence for hfB4, an IRES-Neo selectable marker,and a synthetic polyA. The entire construct was sequenced to confirm themutation and the integrity of the construct. The expected amino acidsequence for hfB4 is shown in SEQ ID NO:17. The only difference betweenthe amino acid sequence of hfB3 and hfB4 is the D740N change.

A stable cell line that expresses hfB4 protein was generated byPEI-mediated transfection and drug selection of 293 cells as describedin Example 3. The concentration of hfB4 protein in the cell culturemedium of the selected cell population was measured by ECL as describedin Example 5.

Biological activity of hfB4 protein, purified as described in Example 9using the one HIC chromatography step method, was examined by hemolyticactivity assay as described in Example 7. Table 2 shows the inhibitionof human alternative complement pathway hemolytic activity by cellculture medium containing hfB4 protein. Relative hemolytic activity wasscored by hemoglobin released after hemolysis of rRBC by humanalternative complement pathway activity. As shown in Table 2, hfB4protein efficiently inhibited the alternative complement pathwayactivity.

TABLE 2 hfB4 inhibition of human alternative complement pathwayhemolytic activity Control w/o wt Competed with 0.5 ug wt hfB hfB4 0.0ug 1.0 ug 0.5 ug 0.25 ug 0.125 ug % Inhibition 100 ± 0.0 99.3 ± 1.5100.3 ± 0.0 100.1 ± 0.3 96.1 ± 0.8

Example 12. Generation and Characterization of an hfB3-Fc ExpressionConstruct and an hfB3-Fc Protein

hfB3-Fc is a fusion protein between hfB3 and an IgG Fc. Specifically,the full length of the hfB3 protein was fused with a human IgG4 Fc.

To create an hfB3-Fc expression construct, a PCR product was amplifiedfrom the hfB3 expression construct (SEQ ID NO:5) described in Example 1using two primers: the forward primer5′-GCGCACCGGTGCTAGCGAATTCGGCGACAAGAAGGGCAGCTGCGA-3′ (SEQ ID NO:19); andthe reverse primer 5′-GCGCAGATCTCAGGAAGCCCAGGTCCTCAT-3′ (SEQ ID NO:20).The 377 bp PCR product containing the coding region for the C-terminusof hfB3-Fc was then digested with Age I and Bgl II and ligated intopFUSE-hIgG4Fc (Invivogen, Cat. Code: pfuse-hg4fc1) which was previouslydigested with Age I and Bgl II, creating the plasmid phfB3Cterm-Fc. ThehfB3 expression construct (SEQ ID NO:5, described in Example 1) wasdigested with EcoR I and EcoR V. The EcoR I/EcoR V fragment containingthe N-terminus of hfB3 was ligated into phfB3Cterm-Fc which waspreviously digested with EcoR I and EcoR V, creating phfB3-Fc. ThephfB3-Fc plasmid was digested with Nhe I and the fragment containing thehfB3 and Fc coding sequences was ligated into the modified pCI constructwith IRES-Neo described in Example 1, creating the hfB3-Fc expressionconstruct (SEQ ID NO:18). This hfB3-Fc expression construct includes,from 5′ to 3′, a CMV promoter, a chimeric intron, a coding sequence forhfB3-Fc, an IRES-Neo selectable marker, and a synthetic polyA. Theentire construct was sequenced to confirm the integrity of the construct(SEQ ID NO:18). SEQ ID NO:21 is the amino acid sequence of the hfB3-Fcprotein.

A stable cell line that expresses hfB3-Fc protein was generated byPEI-mediated transfection and drug selection of 293 cells as describedin Example 3. The drug selected cells were cultured at 2×10⁶ cells/mLfor 72 hours. Then hfB3-Fc protein expression was examined by subjecting2 μl of the cell culture supernatant to a non-reducing SDS-PAGE andWestern blot analysis. As shown in FIG. 8, two bands of hfB3-Fc proteinwere detected by a goat anti-factor B specific antibody (R&D Systems,Cat. No. AF2739). The molecular weight markers in KDa are indicated onthe left. Not wishing to be bound by theory, these two bands of hfB3-Fcprotein might represent monomers and dimers of the protein. Biologicalactivity of hfB3-Fc protein (in cell culture supernatant) was examinedby a hemolytic activity assay as described in Example 7. As shown inTable 3, hfB3-Fc protein inhibited the alternative complement pathwayactivity.

TABLE 3 hfB3-Fc inhibition of human alternative complement pathwayhemolytic activity Control w/o wt compete with 0.5 ug wt hfB Sup. ofhfB3-Fc 0 35 ul 20 ul % inhibition 100 ± 0.0 100.2 ± 0.8 92.6 ± 8.5

Example 13. Effect of Repeated Freeze/Thaw on hfB3-292S ComplementInhibition

hfB3-2925 protein was purified as described in Example 9 using the oneHIC chromatography step method. Purified hfB3-2925 protein in PBS,removed from a −80° C. freezer and thawed at room temperature, wascounted as the first freeze and thaw cycle. After this thaw, thehfB3-2925 protein concentration was adjusted to 2 mg/mL with PBS and onealiquot of hfB3-2925 was sampled and saved on ice as the firstfreeze-and thaw sample. The tube of hfB3-2925 protein was then frozen bysitting the tube in a methanol/dry ice bath for 20 minutes and thenthawing at room temperature till completely thawed (the second freezeand thaw cycle). One aliquot of the sample was sampled and set asidebefore repeating the next freeze and thaw cycle. The biological activityof samples from each cycle of freeze and thaw (total 7 times) wereanalyzed by measuring their ability to compete with wild type humanfactor B in the alternative complement pathway mediated hemolytic assayas described in Example 7. Each reaction contained a fixed amount ofwild type human factor B (0.5 μg) with increasing amounts of hfB3-292S.The results in Table 4 represent the percentage of inhibition in thehemolytic assay.

These results demonstrate that hfB3-292S protein can still effectivelyinhibit alternative pathway mediated hemolysis even after seven cyclesof freeze and thaw (Table 4). A similar level of hemolysis inhibitionwas observed through out all samples showing that repeated freeze andthaw (up to 7 times) did not affect the hfB3-292S protein's ability toinhibit complement mediated hemolysis.

TABLE 4 hfB3-292S After Freeze/Thaw cycles Amount of hfB3-292S in FreezeThaw Cycles Each Reaction 1 2 3 4 5 6 7 1.0 μg 103.6 +/− 0.4 103.9 +/−1.3 103.3 +/− 0.1 100.6 +/− 0.5 101.3 +/− 1.4 102.5 +/− 1.2 101.2 +/−0.4 0.5 μg 104.5 +/− 0.6 102.7 +/− 1.0 102.6 +/− 0.5 101.5 +/− 1.5 102.9+/− 2.1 102.4 +/− 0.9 100.0 +/− 1.8 0.25 μg  98.8 +/− 1.0  99.8 +/− 2.0 99.5 +/− 0.6  97.9 +/− 1.5  97.1 +/− 0.8  97.8 +/− 0.5 95.5 +/− 0.60.125 μg  80.7 +/−2.2  88.3 +/− 1.3  84.2 +/− 1.1  85.6 +/− 0.8  77.6+/− 3.7  80.0 +/− 1.4 74.8 +/− 2.3

Example 14. Greater Thermostability of hfB3-292S Protein than hfB3Protein

hfB3 and hfB3-292S proteins were purified as described in Example 9using the one HIC chromatography step method. Purified hfB3 andhfB3-292S protein, both in PBS, were removed from −80° C. Proteinconcentration was re-adjusted to 2 mg/mL in PBS (pH 7.4). hfB3 andhfB3-2925 proteins were equally aliquoted into three 0.6 mL eppendorftubes (40 μL per tube) and then stored at 4° C., −80° C. and 37° C.conditions for 7 days. The biological activity (ability to inhibitcomplement mediated hemolysis) of each of the stored samples wasanalyzed by measuring their ability to inhibit alternative complementpathway mediated hemolysis as described in Example 7. The results ofthis hemolytic assay showed that storage at 4° C. or −80° C. over 7 daysdid not affect the ability of either hfB3 or hfB3-292S to inhibitalternative complement pathway mediated hemolysis. However, hfB3 proteinstored at 37° C. for 7 days lost essentially all of its biologicalactivity in all four samples tested. Remarkably, hfB3-292S stored at 37°C. for 7 days preserved its biological activity well and still couldcompete with wild-type human factor B effectively (Table 5). The resultsin the Table 5 represent the percentage of inhibition of humanalternative complement activity (with standard deviation) by either hfB3or hfB3-292S. These results indicated that hfB3-292S protein has greaterthermostability than hfB3 protein at 37° C.

TABLE 5 Thermostability of hfB3 and hfB3-292S Sample Treatment 1.0 μg0.5 μg 0.25 μg 0.125 μg Amount of Purified hfB3 in Each Reaction + 0.5μg Wild Type Human Factor B hfB3 4° C. for 99.6 ± 0.2 99.1 ± 0.9 98.2 ±0.6 88.7 ± 0.7 7 days −80° C. for 99.8 ± 1.2 98.2 ± 1.8 97.2 ± 1.1 89.2± 5.8 7 days 37° C. for  0.0 ± 5.5  0.0 ± 3.7  0.3 ± 5.8  4.1 ± 6.4 7days Amount of Purified hfB3-292S in Each Reaction + 0.5 μg Wild TypeHuman Factor B hfB3-292S 4° C. for 98.4 ± 0.6 98.4 ± 0.9 97.8 ± 0.3 83.1± 3.0 7 days −80° C. for 99.0 ± 0.3 98.4 ± 0.7 96.8 ± 0.8 81.7 ± 2.1 7days 37° C. for 98.2 ± 0.4 94.6 ± 0.9 78.2 ± 2.9 48.0 ± 1.3 7 days

Example 15. Protein Melting Point Determination of Human Factor B, fB3,and fB3-292S Proteins

Protein melting temperature (Tm) is a measure of the thermal stabilityof a protein and changes in the amino acid sequence of a protein mayaffect, among other things, the protein's thermal stability. Humanfactor B protein (hfB) contains twenty-three cysteine residues,twenty-two of which occur as disulfide bond pairs (cystine) and one ofwhich, C292, is present in an unpaired free sulfhydrile form.

The melting temperature profiles of hfB3 protein (K258A, R259A, K260A,D279G, N285D) and hfB3-292S protein (the single unpaired cysteineresidue of hfB3 protein was modified to serine) were compared to that ofhfB protein by incubating each protein in the presence of1-anilinonapthalene-8-sulfonic acid (ANS) and measuring the increase influorescence of ANS at 460 nm. ANS binds to protein hydrophobic regions(Stryer, J. Molecular Biology (1965) 13:482-495) and has been used toinvestigate the effect of temperature on the surface hydrophobicity ofhfB protein (Takada, et. al., Complement (1985) 2:193-203). Samplescontaining hfB protein (35 μg), hfB3 protein (50 μg) and hfB3-2925protein (39 μg) were prepared in 100 μL of PBS (137 mM NaCl, 2.7 mM KCl,10 mM phosphate, pH 7.4) buffer containing 10 mM ANS (Invitrogen,Catalog # A-47). Samples of each protein (in triplicate) were incubatedin closed polypropylene tubes for thirty minutes at 21° C., 30° C., 37°C., 44° C., 47° C., 50° C., 55° C., 60° C., and 65° C. The samples weretransferred to a clear 96-well microplate (Costar, Catalog #3635) andthe fluorescence was measured in a Perceptive Biosystems Cytofluor 4000microplate spectrofluorometer (excitation=60/40 nm, emission=460/40 nm).The results were analyzed using a non-linear 4PL curve fit. The Tmvalues (average of the results from two experiments) for wild type hfBprotein, hfB3 protein and hfB3-2925 protein were determined to be 46.4°C., 45.1° C., and 47.0° C., respectively. The five amino acid changesmade in hfB3 protein with respect to wild type hfB protein (K258A,R259A, K260A, D279G, N285D) resulted in a ΔTm=−1.3° C., indicating thathfB3 protein was less thermally stable compared to a corresponding wildtype hfB protein. However, the Tm of hfB3-2925 protein (47.0° C.)resulted in a ΔTm=+1.9° C. with respect to hfB3 protein and indicatedthat hfB3-2925 protein was at least as thermally stable as wild type hfBprotein (46.4° C.).

Therefore, contrary to the results of Culajay, et. al. (Biochemistry(2000) 39:7153-7158) which showed that substitution of a cysteineresidue with serine in human fibroblast growth factor (FGF-1) protein(C83S or C117S) decreased the Tm by 13° C. and 2° C., respectively, theserine substitution of hfB3 at amino acid C292 resulted in hfB3-2925being more thermal stable than hfB3.

Example 16. hfB3-292S Protein Prevents Joint Inflammation and Damage ina Mouse Rheumatoid Arthritis Model

Collagen antibody-induced arthritis (CAIA) is an aggressive mouse modelfor rheumatoid arthritis (Terato K et al., J. Immunol. (1992)148(7):2103-8; Terato K et al., Autoimmunity (1995) 22(3):137-47). Inthis model, a collagen antibody cocktail containing 4 monoclonalantibodies against collagen (Chondrex, Inc., Catalogue Number: 10010)with LPS boost was used to induce arthritis in six-week-old DBA/1J malewild type mice (Jackson Laboratory).

Forty mice were divided into three groups. Group 1 had 10 mice servingas a vehicle control group where 100 μL PBS (phosphate buffered saline,pH 7.4) was injected into the tail vein on day 0, a booster injection of25 μg LPS (List Biological Lab, Campbell, Calif., Catalogue Number: 421)per mouse was administrated intraperitoneal (LPS was in PBS at aconcentration 500 μg/mL) on day 3, and 100 μL of PBS via the tail veinagain on days 3, 5, 7, and 9. Group 2 had 15 mice that were injectedwith the collagen antibody cocktail (0.25 mg in 100 μL PBS per mouse)via tail vein on day 0, received a booster injection of 25 μg LPS on day3, and were administered 100 μL PBS via the tail vein on days 3, 5, 7,and 9. Group 3 had 15 mice that were injected with 0.25 mg of thecollagen antibody cocktail and 1 mg of hfB3-2925 protein, both togetherin 100 μL PBS per mouse via the tail vein on day 0, administered abooster injection of 25 μg LPS on day 3, and administered 1 mg ofhfB3-2925 protein in 100 μL PBS via tail vein on days 3, 5, 7, and 9.

Mice were examined every day. Each mouse weight was recorded when jointmeasurement took place. Forepaws and hind limb joints were measuredusing calipers for both width and thickness on days −1, 4, 6, 9 and 11.The measuring sequence was left front limb, left hind limb, right hindlimb and right front limb. On day 11, all animals were sacrificed andall the limbs (left front limb, right front limb, left hind limb andright hind limb) were collected and stored in individually-labeledplastic cassettes. Each cassette was placed in a histology container boxcontaining 10% neutral buffered formalin solution.

These mouse limbs were subjected to paraffin sectioning and H&E stainingto examine the pathogenesis in the joints. Specifically, followingfixation, each limb from each mouse was transferred to a plasticcassette separately. The limbs were rinsed with running water in abeaker for 30 minutes (min) at room temperature (RT) to remove fixativesolution. Then each cassette was transferred to a beaker containingdecalcified solution (Thermo Scientific, Catalogue number 8340) byimmersion of the cassette into the solution. Front limbs weredecalcified for 8 hours and hind limbs were decalcified for 9 hours.After 8 hours or 9 hours, the limbs were again rinsed with running waterfor 30 min at RT to remove decalcified solution. After decalcification,limbs were stored in 70% ethanol overnight (O/N) for the nextdehydration step. Limbs were dehydrated by sequentially immersing into:75% ethanol (made from 100% ethanol) for two times; 85% ethanol for twotimes, 95% ethanol for two times and 100% ethanol for two times, eachtime for 15 min at RT with shaking. Next, the limbs were immersed in a1:1 mixture of 100% ethanol and cedar wood oil (Fisher, CatalogueNumber: 040-1) for 15 min at RT with shaking, and repeated two moretimes for a total of three times. The limbs were then immersed in 100%cedar wood oil and incubated at 40° C. for 5 hrs. Following the 5 hourincubation, limbs were immersed in a 1:1 mixture of cedar wood oil andmethyl salicylate (ACROS, Catalogue number 119-36-8) for 60 min at RT.The limbs were then immersed in another 1:1 mixture of cedar wood oiland methyl salicylate O/N at RT. Following the O/N incubation, limbswere immersed in 100% methyl salicylate for 40 min at RT, repeated onemore time (a total of two times). Finally, limbs were embedded inparaffin that was prepared by incubation at 60° C. for 7 hrs.

Paraffin sections of the mouse limbs were prepared using a microtome andcut to a 7 μm thickness. The sections were incubated on a 40° C. waterbath and transferred to a Superfrost Plus microscope slide. The slideswere dried O/N at RT, and further dried by incubation O/N on a slidewarmer. The slides were kept at RT until staining.

Mounted paraffin sections of the mouse limb were subjected to H&Estaining. The sections were de-paraffinized and rehydrated by immersioninto xylene for 3 min repeated 2 times for a total of 3 times. Theexcess xylene was then blotted, and sections were immersed in 100%ethanol for 3 min, for a total of 3 times, then 95% ethanol for 3 min,once, 80% ethanol for 3 min, once, and deionized water for 5 min, once.All incubations were at RT. For the hematoxylin staining, slides wereimmersed in hematoxylin for 4 min, one time, and rinsed with deionizedwater. The slides were immersed in tap water for 5 min one time to allowthe stain to develop. The slides were dipped quickly, 8-12 times, intoacid ethanol (200 ml 70% ethanol plus 150 μL concentrated HCL) todestain the sections. The slides were then rinsed twice for 1 min in tapwater, and then once for 2 min in deionized water. The excess water wasblotted from the slides prior to eosin staining.

For the eosin staining, slides were immersed in eosin once for 20seconds. Slides were then dehydrated by immersion into 95% ethanol 3times for 5 min. Slides were incubated in 100% ethanol 3 times for 5min. The excess ethanol was blotted, and the slides were incubated inxylene three times for 15 min. Coverslips were adhered to the slidesusing the xylene-based Permount (EMS, Catalogue number 17986-01) byplacing a drop of Permount on the slide using a glass rod being carefulnot to form bubbles. The coverslip was then angled onto the slide anddropped gently onto the slide. The Permount was allowed to spreadbeneath the coverslip covering the entire section. The slides were driedO/N at RT in a chemical hood.

As shown in FIG. 10, the group injected with collagen antibody cocktailin the absence of hfB3-292S (Group 2) induced severe front paw swelling.Mice injected with the antibody cocktail, but were treated withhfB3-292S protein (Group 3) showed a 65% reduction in the size of thepaw (p<0.0003) (FIG. 10). These results demonstrate a significantinhibitory effect of joint arthritis in this model by hfB3-292S. At 250μg collagen antibody cocktail dosage per mouse for the induction ofCAIA, the hind limbs of the mice did not show obvious swelling. Nosignificant adverse effect for hfB3-292S protein treatment on mouseweight was observed. The average weight of all mice maintained steadily.The average weight for all mice in all three groups was 20.4±1.1 gramsby the end of the study.

As shown in FIG. 9, the mice in Group 1 appeared to have normal joints,no detectable inflammatory cell infiltration into the joint and thecartilage and bones appeared normal (FIG. 9, top panel). The mice inGroup 2 had severe inflammation in the joints, inflammatory cellinfiltration, pannus formation, cartilage damage and bone erosion (FIG.9, middle panel). The mice in Group 3 treated with hfB3-292S had normaljoint structure, no inflammatory cell infiltration, no cartilage or boneerosion or damage (FIG. 9, bottom panel).

These data demonstrated that hfB3-292S was significantly efficacious inpreventing joint inflammation and damage in this CAIA mouse model,demonstrating the therapeutic utility of hfB3-292S protein forrheumatoid arthritis.

Example 17. Generation and Characterization of an hfB3-292S-FcExpression Construct and an hfB3-292S-Fc Protein

A stable cell line that expresses hfB3-292S-Fc protein (SEQ ID NO:22)was generated by PEI-mediated transfection and drug selection of 293cells as described in Example 3. The drug selected cells were culturedat 2×10⁶ cells/mL for 72 hours. Then hfB3-2925-Fc protein expression wasexamined by subjecting 2 μL of the cell culture supernatant to anon-reducing SDS-PAGE and Western blot analysis. Two bands ofhfB3-2925-Fc protein were detected by a goat anti-factor B specificantibody. (Data not shown.) Not wishing to be bound by theory, these twobands of hfB3-2925-Fc protein might represent monomers and dimers of theprotein.

hfB3-292S-Fc was purified with a Protein A column. Biological activityof purified hfB3-292S-Fc protein was examined by a hemolytic activityassay as described in Example 7. As shown in Table 6, hfB3-292S-Fcprotein inhibited the alternative complement pathway activity in a dosedependent manner.

TABLE 6 hfB3-292S-Fc inhibition of human alternative complement pathwayhemolytic activity Amount of Control w/o wt hfB Compete with 0.5 μg wthfB hfB3-292S-Fc (μg) 0 2.0 1.0 0.5 0.3 % inhibition 100 +/− 0.0 93.8+/− 1.9 75.4 +/− 1.2 47.3 +/− 3.2 34.0 +/− 5.3

Example 18. C-Terminus Truncated hfB3-292S

A gene expression construct was made that expressed a truncated form ofhfB3-292S with the C-terminal 284 amino acids (the serine proteasedomain) being deleted. The molecule is designated as hfB3-292SN480 whichis made up of the N-terminal 480 amino acids of hfB3-292S (amino acids1-480 of SEQ ID NO:2 or amino acids 26-480 of SEQ ID NO:2 after cleavageof the secretion peptide). The DNA sequence of the expression constructfor hfB3-292SN480 is shown in SEQ ID NO:24 with nucleotides 1064-2509being the coding sequence for hfB3-292SN480. The expression constructwas transfected into 293 FreeStyle cells and selected with G418 asdescribed previously in Example 3. The G418 resistant non-clonal cellculture medium was subjected to Western blot analysis for hfB3-292SN480expression using full-length hfB3-2925 as a control (left lane). Asshown in FIG. 11, the Western blot analysis with a monoclonal antibodyspecifically for hfB3-2925 detected a band approximately 55 KDa from thecell culture medium of hfB3-292SN480 cell line (right lane), suggestingthat even with a 280 amino acid deletion from the C-terminus ofhfB3-2925, the N-terminal 480 amino acids can be expressed at anappropriate size.

An alternative complement activity assay was performed as describedpreviously, to determine if hfB3-292SN480 can inhibit alternativecomplement activity. As shown in FIG. 13 the cell culture supernatant,from cells expressing hfB3-292SN480, inhibited the alternativecomplement activity in a dose-dependent manner. This demonstrates thatfragments of hfB3-292S can still retain the ability to inhibitcomplement activity and therefore can be utilized the same as describedherein for hfB3-292S (SEQ ID NO:2)

Example 19. Monomeric hfB3-292S/Fc Fusion Protein hfB3-292S/Fc-Mono

A gene expression construct encoding a full-length hfB3-2925 “fused” toa human IgG4 Fc was engineered. hfB3-2925 is a monomer when it isproduced in mammalian cells, such as human cells as describedpreviously, e.g., see FIG. 3 described in Example 6 which showshfB3-2925 was detected as one band at approximately MW 100 KDa undernon-reducing conditions, suggesting hfB3-2925 is a monomer. Twocysteines in the hinge region of the human IgG4 Fc were mutated toensure the fusion protein would be monomer and retain hfB3-292S'sbiological property for inhibiting complement activity. The twocysteines were mutated by substituting them each with a serine. Thisfusion protein of hfB3-2925 and the mutated IgG4 Fc was designatedhfB3-2925/Fc-mono. The DNA sequence for this fusion protein expressionconstruct is shown in SEQ ID NO:26. The corresponding amino acidsequence for hfB3 2925/Fc mono is shown in SEQ ID NO:25 with amino acids1-764 being the hfB3-2925 region and amino acids 765-1003 being thehuman IgG4 Fc region with amino acids 782 and 785 being serine aminoacids that were substituted for cysteine residues found in a nativehuman IgG4 Fc.

The hfB3-2925/Fc-mono gene expression construct (SEQ ID NO:26) wastransfected into human 293 FreeStyle cells. The cells were thensubjected to G418 selection. The culture medium from the drug resistantcells was subjected to Western blot analysis for the fusion protein. Asshown in FIG. 12, a band at approximately 115 KDa was detected bypurified goat anti-human factor B antibody in this non-reducing SDS-PAGEand Western blot analysis. The two higher bands most likely wereaggregates of the monomeric fusion protein. The data suggested that themonomeric fusion protein between hfB3-2925 and human IgG4 Fc(hfB3-2925/Fc-mono) was successfully expressed in mammalian cells andthat the majority of the fusion protein appeared to be monomeric.

To examine if hfB3-2925/Fc-mono preserved hfB3-2925's property ofblocking alternative complement activity, an alternative complementactivity assay was performed as described previously. As shown in FIG.14, the cell culture supernatant from hfB3-2925/Fc-mono producing cellsinhibited the alternative complement activity in a dose-dependentmanner, suggesting that hfB3-2925's complement inhibitory activity wasnot lost in this monomeric hfB3-2925/Fc-mono fusion protein.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference in their entiretyinto the specification to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference.

What is claimed is:
 1. A method of inhibiting activity of thealternative complement pathway, wherein the method comprises introducingor administering to a site of the complement activity a polypeptidecomprising a human complement factor B protein analog, wherein the humancomplement factor B analog comprises a substitution of a free cysteineamino acid corresponding to amino acid 292 of SEQ ID NO:1 as determinedby alignment of the amino acid sequence of the human complement factor Banalog with the amino acid sequence of SEQ ID NO: 1; and the humancomplement factor B protein analog is at least 90% identical to aminoacids 26-764 of SEQ ID NOs:1, 2 or 3; to amino acids 26-990 of SEQ IDNOs:22 or 23; to amino acids 26-480 of SEQ ID NO:2; or to amino acids26-1003 of SEQ ID NO:26.
 2. The method of claim 1, wherein the freecysteine is substituted with an amino acid selected from the groupconsisting of alanine, histidine, isoleucine, leucine, methionine,phenylalanine, serine, threonine, tyrosine and valine.
 3. The method ofclaim 1, wherein the complement factor B protein analog furthercomprises mutations corresponding to K258A, R259A, K260A, D279G andN285D of SEQ ID NO:1.
 4. The method of claim 1, wherein the complementfactor B protein analog comprises a mutation in the C3b binding domainand the complement factor B protein analog exhibits increased bindingaffinity to C3b as compared to the binding affinity of a correspondingnative complement factor B protein to C3b.
 5. The method of claim 4,wherein the mutation in the C3b binding domain comprises: (i) asubstitution or deletion of an aspartic acid corresponding to amino acid279 of SEQ ID NO:1, a substitution or deletion of an asparaginecorresponding to amino acid 285 of SEQ ID NO:1 or both; or (ii) aninsertion of at least one amino acid next to said aspartic acid or saidasparagine.
 6. The method of claim 1, wherein the complement factor Bprotein analog comprises amino acids 26-480 of SEQ ID NO:2.
 7. Themethod of claim 6, wherein the polypeptide consists of amino acids26-480 of SEQ ID NO:2.
 8. The method of claim 1, wherein the complementfactor B protein analog comprises amino acids 26-764 of SEQ ID NO:2. 9.The method of claim 8, wherein the polypeptide consists of amino acids26-764 of SEQ ID NO:2.
 10. The method of claim 1, wherein thepolypeptide consists of SEQ ID NO:2.
 11. The method of claim 1, whereinthe polypeptide consists of SEQ ID NO:3.
 12. The method of claim 1,wherein the polypeptide consists of SEQ ID NO:4.
 13. The method of claim1, wherein the polypeptide comprises an immunoglobulin Fc domain. 14.The method of claim 13, wherein the polypeptide consists of SEQ IDNO:22.
 15. The method of claim 13, wherein the polypeptide consists ofSEQ ID NO:23.
 16. The method of claim 13, wherein the polypeptideconsists of SEQ ID NO:24.
 17. The method of claim 13, wherein thepolypeptide consists of SEQ ID NO:26.
 18. The method of claim 1, whereinthe complement factor B protein analog exhibits increased bindingaffinity to factor D as compared to the binding affinity of acorresponding native complement factor B protein to factor D.
 19. Themethod of claim 1, wherein the administration is administration to apatient having an alternative complement pathway-mediated disease. 20.The method of claim 19, wherein the alternative complementpathway-mediated disease is a disease of the eye.
 21. The method ofclaim 20, wherein the polypeptide is administered to the eye.
 22. Themethod of claim 19, wherein the alternative complement pathway-mediateddisease is macular degeneration, age-related macular degeneration (AMD),geographic atrophy, wet AMD, myocardial infarction, dry AMD, drusenformation, arthritis, stroke, ischemic reperfusion injury, diabeticretinopathy, vitreoretinopathy, traumatic organ injury, cornealinflammation, corneal neovascularization, uveitis, ocular hypertensionor glaucoma.
 23. The method of claim 19, wherein the alternativecomplement pathway-mediated disease is selected from the groupconsisting of atherosclerosis, airway hyperresponsiveness, immunerelated diseases, autoimmune related diseases, lupus nephritis, systemiclupus erythematosus (SLE), arthritis, rheumatologic diseases,anti-phospholipid antibody syndrome, intestinal and renal I/R injury,asthma, atypical hemolytic-uremic syndrome, Type IImembranoproliferative glomerulonephritis, non-proliferativeglomerulonephritis, fetal loss, brain injury, post-traumatic organdamage, post infarction organ damage, vasculitis, hereditary angioedema,paroxysmal nocturnal hemoglobinuria, cerebrovascular accident,Alzheimer's disease, transplant rejection, infections, sepsis, septicshock, Sjögren's syndrome, myasthenia gravis, antibody-mediated skindiseases, Type I and Type II diabetes mellitus, insulin resistancesyndrome, gestational diabetes, thyroiditis, idiopathic thrombocytopenicpurpura and hemolytic anemia, neuropathies, multiple sclerosis,cardiopulmonary bypass injury, polyarteritis nodosa, Henoch Schonleinpurpura, serum sickness, Goodpasture's disease, systemic necrotizingvasculitis, post streptococcal glomerulonephritis, idiopathic pulmonaryfibrosis, membranous glomerulonephritis, acute shock lung syndrome,adult respiratory distress syndrome, and reperfusion.